INSULIN-LIKE GROWTH FACTOR BINDING PROTEIN, ACID LABILE SUBUNIT (IGFALS) AND INSULIN-LIKE GROWTH FACTOR 1 (IGF-1) iRNA COMPOSITIONS AND METHODS OF USE THEREOF

ABSTRACT

The present invention relates to RNAi agents, e.g., double stranded RNAi agents, targeting the insulin-like growth factor binding protein, acid labile subunit (IGFALS) gene or the insulin-like growth factor 1 (IGF-1) gene, methods of using such double stranded RNAi agents to inhibit expression of an IGFALS gene or an IGF-1 gene, and methods of treating subjects having an IGF system-associated disorder.

RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No. 16/601,681, filed on Oct. 15, 2019, which is a divisional of U.S. patent application Ser. No. 15/743,349, filed on Jan. 10, 2018, now U.S. Pat. No. 10,494,632, issued on Dec. 3, 2019, which is a 35 U.S.C. § 371 national stage filing of International Application No. PCT/US2016/041440, filed on Jul. 8, 2016, which in turn claims the benefit of priority to U.S. Provisional Patent Application No. 62/191,008, filed on Jul. 10, 2015, U.S. Provisional Patent Application No. 62/269,401, filed on Dec. 18, 2015, and U.S. Provisional Patent Application No. 62/316,726, filed on Apr. 1, 2016. The entire contents of each of the foregoing applications are hereby incorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy, created on May 14, 2021, is named 121301-03905_SL.txt and is 1,164,983 bytes in size.

BACKGROUND OF THE INVENTION

Acromegaly is a progressive and life threatening disease resulting from growth hormone hypersecretion from a benign pituitary tumor, leading to approximately a 10 year reduction in lifespan and a reduced quality of life. Acromegaly is associated with cardiovascular disease including hypertension and cardiac hypertrophy, cerebrovascular disease including stroke, metabolic disease including diabetes, and respiratory disease including sleep apnea. Mortality rates in acromegaly are correlated with growth hormone and IGF-1 levels, with increased growth hormone concentrations being associated with shorter life spans (Holdaway et al., JCEM, 2004). The clinical features most commonly associated with acromegaly are acral enlargement, maxofacial changes, excessive sweating, athralgias, headache, hypogonadal symptoms, visual deficit, fatigue, weight gain, and galactorrhea. Such symptoms may be associated with any of a number of diseases or conditions and, thus, diagnosis of acromegaly often does not occur until several years after the initiation of growth hormone hypersecretion. Definitive diagnosis of acromeagly includes detection of an increased level of insulin-like growth factor-1 (IGF-1) and growth hormone elevation in an oral glucose tolerance test, confirmed by detection of a GH-hypersecreting pituitary tumor, typically by MRI. (The diagnostic criteria for acromegaly are provided in the American Association of Clinical Endocrinologists Medical Guidelines for Clinical Practice for the Diagnosis and Treatment of Acromegaly—2011 Update (Katznelson et al., Endocr. Pract. 17 (Suppl. 4)).

Current treatment options for acromegaly are insufficient for many patients. Surgical removal of the pituitary adenoma by transsphenoidal surgery results in a cure for about 50-60% of patients. Subjects for whom surgical intervention is not possible or does not result in a cure are treated with first-line pharmacological therapy which includes dopamine agonists or sustained-release somatostatin analogs (SSAs). This therapy results in good control for the disease for about 70% of these patients for whom surgery cannot provide a cure. The use of SSAs, however, is limited to subjects expressing a somatostatin receptor on their tumor. Subjects whose disease cannot be controlled by the first-line pharmacological therapy are treated with SOMAVERT® (pegvisomant), a growth hormone receptor antagonist, which is administered by daily subcutaneous injection. Radiotherapy, which suffers from low efficacy and high side effects, is used as a last resort.

The insulin-like growth factor system is also associated with abnormal growth in cancer and metastasis (see, e.g., Samani et al., Endocrine Rev., 2007). The IGF system has become a target for anticancer agents, both as primary and adjunctive therapy.

Currently, treatments for acromegaly and cancer do not fully meet patient needs. Therefore, there is a need for therapies for subjects suffering from acromegaly or cancer.

SUMMARY OF THE INVENTION

The present invention provides iRNA compositions which affect the RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of an insulin-like growth factor binding protein, acid labile subunit (IGFALS) gene or an insulin-like growth factor-1(IGF-1) gene. The IGFALS gene or IGF-1 gene may be within a cell, e.g., a cell within a subject, such as a human.

In an aspect, the invention provides a double stranded ribonucleic acid interference (dsRNA) agent for inhibiting expression of insulin-like growth factor binding protein, acid labile subunit (IGFALS), wherein the double stranded dsRNA agent comprises a sense strand and an antisense strand, wherein the sense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:1 and the antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:2.

In certain embodiments, the sense strands and antisense strands comprise sequences selected from any one of the sequences in any one of Tables 3, 5, 6, 8, 12, or 14.

In an aspect, the invention provides a double stranded ribonucleic acid interference (dsRNAi) agent for inhibiting expression of insulin-like growth factor-1 (IGF-1), wherein the double stranded RNAi agent comprises a sense strand and an antisense strand, wherein the sense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO: 11 or 13 and the antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO: 12 or 14.

In certain embodiments, the sense strands and antisense strands comprise sequences selected from any one of the sequences in any one of Tables 9, 11, 15, 17, 18, or 20.

In an aspect, the invention provides a double stranded ribonucleic acid interference (dsRNAi) agent for inhibiting expression of insulin-like growth factor binding protein, acid labile subunit (IGFALS), wherein the double stranded RNAi comprises a sense strand and an antisense strand, the antisense strand comprising a region of complementarity which comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from any one of the antisense sequences listed in any one of Tables 3, 5, 6, 8, 12, or 14.

In an aspect, the invention provides a double stranded ribonucleic acid interference (dsRNAi) agent for inhibiting expression of insulin-like growth factor 1 (IGF-1) wherein the double stranded RNAi comprises a sense strand and an antisense strand, the antisense strand comprising a region of complementarity which comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from any one of the antisense sequences listed in any one of Tables 9, 11, 15, 17, 18, or 20.

In certain embodiments, the double stranded RNAi comprises at least one modified nucleotide. In some embodiments, substantially all of the nucleotides of the sense strand are modified nucleotides. In some embodiments, substantially all of the nucleotides of the antisense strand are modified nucleotides. In some embodiments, all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand comprise a modification.

In an aspect, the invention provides a double stranded RNAi agent for inhibiting expression of insulin-like growth factor binding protein, acid labile subunit (IGFALS), wherein the double stranded RNAi agent comprises a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:1 and the antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:2, wherein substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides, and wherein the double stranded RNAi agent comprises a ligand, e.g., the sense strand of the double stranded RNAi agent is conjugated to a ligand, e.g., a ligand is attached at the 3′-terminus of the sense strand.

In an aspect, the invention provides a double stranded ribonucleic acid (RNAi) agent for inhibiting expression of insulin-like growth factor 1 (IGF-1), wherein the double stranded RNAi agent comprises a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO: 11 or 13 and the antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO: 12 or 14, wherein substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides, and wherein the sense strand is conjugated to a ligand attached at the 3′-terminus.

Accordingly, in certain embodiments, the present invention provides double stranded RNAi agents for inhibiting expression of IGFALS, which comprise a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from nucleotides 11-62, 24-62, 79-117, 79-130, 155-173, 194-216, 194-229, 211-229, 232-293, 254-272, 310-328, 310-349, 324-345, 331-349, 353-371, 353-394, 376-394, 407-425, 439-449, 431-470, 484-515, 497-515, 541-580, 547-568, 596-647, 616-634, 673-691, 694-712, 694-734, 777-799, 781-799, 825-843, 825-855, 869-922, 958-976, 958-988, 1064-1085, 1064-1096, 1067-1085, 1067-1096, 1100-1141, 1111-1129, 1145-1163, 1145-1186, 1159-1186, 1168-1196, 1168-1214, 1193-1214, 1266-1307, 1321-1339, 1342-1373, 1375-1406, 1432-1450, 1454-1472, 1519-1537, 1519-1559, 1534-1555, 1541-1559, 1606-1624, 1606-1637, 1613-1635, 1672-1690, 1672-1712, 1749-1779, 1783-1801, 1805-1823, 1806-1829, 1871-1889, 1871-1919, 1949-1977, 1993-2011, 2013-2042, 2048-2077, 2048-2088, or 2052-2084 of SEQ ID NO: 1, and, in certain embodiments, the antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the corresponding position of the nucleotide sequence of SEQ ID NO: 2 such that the antisense strand is complementary to the at least 15 contiguous nucleotides differing by no more than 3 nucleotides in the sense strand.

Accordingly, in certain embodiments, the present invention provides double stranded RNAi agents for inhibiting expression of insulin-like growth factor 1 (IGF-1), which comprise a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from nucleotides 330-369, 342-369, 432-490, 432-482, 436-462, 534-559, 330-350, 342-362, 348-368, 349-369, 432-452, 435-455, 436-456, 438-458, 440-460, 441-461, 442-462, 449-469, 455-475, 460-480, 461-481, 462-482, 464-484, 470-490, 484-501, 534-554, 536-556, 538-558, 539-559, 542-562, 548-568, 577-597, 582-602, or 640-660 of the nucleotide sequence of SEQ ID NO: 11, and the antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the corresponding position of the nucleotide sequence of SEQ ID NO: 12 such that the antisense strand is complementary to the at least 15 contiguous nucleotides differing by no more than 3 nucleotides in the sense strand.

In certain embodiments, the present invention provides double stranded RNAi agents for inhibiting expression of insulin-like growth factor 1 (IGF-1), which comprise a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from nucleotides 6-90, 127-145, 185-238, 247-265, 277-295, 389-417, 430-480, 543-561, 654-690, 750-768, 774-870, 894-930, 1007-1029, 1075-1126, 1144-1162, 1197-1215, 1232-1250, 1293-1311, 1334-1352, 1388-1458, 1463-1490, 1511-1529, 1599-1617, 1643-1661, 1690-1727, 1793-1825, 1843-1861, 2057-2075, 2090-2130, 2192-2228, 2310-2332, 2357-2375, 2521-2539, 2566-2588, 2648-2684, 2793-2811, 2962-2980, 3120-3142, 3208-3233, 3269-3287, 3417-3435, 3449-3467, 3575-3603, 3686-3704, 3721-3739, 3806-3824, 3939-3957, 3982-4018, 4081-4037, 4154-4172, 4271-4289, 4319-4377, 4436-4478, 4484-4502, 4523-4545, 4566-4584, 4610-4660, 4686-4717, 4734-4769, 4780-4798, 4815-4843, 4884-4902, 4911-4929, 5004-5034, 5050-5068, 5171-5256, 5311-5364, 5409-5430, 5551-5588, 5609-5638, 5694-5712, 5715-5758, 5790-5808, 5906-5928, 5934-5952, 6323-6345, 6399-6417, 6461-6497, 6510-6535, 6584-6612, 6629-6647, 6661-6683, 6726-6789, 6796-6824, 6826-6851, 6858-6905, 6910-6927, 7004-7022, 7035-7130, 7144-7162, 7175-7241, and 7252-7270 of the nucleotide sequence of SEQ ID NO: 13, and the antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the corresponding position of the nucleotide sequence of SEQ ID NO: 14 such that the antisense strand is complementary to the at least 15 contiguous nucleotides differing by no more than 3 nucleotides in the sense strand.

In certain embodiments, the sense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from nucleotides 342-369, 432-462, 330-350, 342-362, 348-368, 349-369, 432-452, 435-455, 436-456, 438-458, 440-460, 442-462, 470-490, 481-501, 536-556, or 539-559 of the nucleotide sequence of SEQ ID NO: 11 and the antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the corresponding position of the nucleotide sequence of SEQ ID NO: 12 such that the antisense strand is complementary to the at least 15 contiguous nucleotides differing by no more than 3 nucleotides in the sense strand. In certain embodiments, the sense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from nucleotides 340-369, 430-490, 430-482, 434-460, 532-559, 328-350, 340-362, 346-368, 347-369, 430-452, 433-455, 434-456, 436-458, 438-460, 439-461, 440-462, 447-469, 453-475, 458-480, 459-481, 460-482, 461-483, 462-484, 468-490, 479-501, 532-554, 534-556, 536-558, 537-559, 540-562, 546-568, 575-597, 580-602, or 638-660 of the nucleotide sequence of SEQ ID NO: 11, for example nucleotides 342-369, 432-462, 330-350, 342-362, 348-368, 349-369, 432-452, 435-455, 436-456, 438-458, 440-460, 442-462, 470-490, 481-501, 536-556, or 539-559 of the nucleotide sequence of SEQ ID NO: 11, and the antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the corresponding position of the nucleotide sequence of SEQ ID NO: 12 such that the antisense strand is complementary to the at least 15 contiguous nucleotides differing by no more than 3 nucleotides in the sense strand.

In certain embodiments, the sense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from nucleotides 6-90, 127-145, 185-238, 247-265, 277-295, 389-417, 430-480, 543-561, 654-690, 750-768, 774-870, 894-930, 1007-1029, 1075-1126, 1144-1162, 1197-1215, 1232-1250, 1293-1311, 1334-1352, 1388-1458, 1463-1490, 1511-1529, 1599-1617, 1643-1661, 1690-1727, 1793-1825, 1843-1861, 2057-2075, 2090-2130, 2192-2228, 2310-2332, 2357-2375, 2521-2539, 2566-2588, 2648-2684, 2793-2811, 2962-2980, 3120-3142, 3208-3233, 3269-3287, 3417-3435, 3449-3467, 3575-3603, 3686-3704, 3721-3739, 3806-3824, 3939-3957, 3982-4018, 4081-4037, 4154-4172, 4271-4289, 4319-4377, 4436-4478, 4484-4502, 4523-4545, 4566-4584, 4610-4660, 4686-4717, 4734-4769, 4780-4798, 4815-4843, 4884-4902, 4911-4929, 5004-5034, 5050-5068, 5171-5256, 5311-5364, 5409-5430, 5551-5588, 5609-5638, 5694-5712, 5715-5758, 5790-5808, 5906-5928, 5934-5952, 6323-6345, 6399-6417, 6461-6497, 6510-6535, 6584-6612, 6629-6647, 6661-6683, 6726-6789, 6796-6824, 6826-6851, 6858-6905, 6910-6927, 7004-7022, 7035-7130, 7144-7162, 7175-7241, or 7252-7270 of the nucleotide sequence of SEQ ID NO: 13, and the antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the corresponding position of the nucleotide sequence of SEQ ID NO: 14 such that the antisense strand is complementary to the at least 15 contiguous nucleotides differing by no more than 3 nucleotides in the sense strand.

In certain embodiments, substantially all of the nucleotides of the sense strand are modified. In certain embodiments, substantially all of the nucleotides of the antisense strand are modified nucleotides. In certain embodiments, substantially all of the nucleotides of both strands are modified. Further, in certain embodiments, the double stranded RNAi agent comprises a ligand, e.g., the double stranded RNAi agent is conjugated to at least one ligand, wherein the ligand is one or more GalNAc derivatives attached through a monovalent, a bivalent or a trivalent branched linker.

In certain embodiments, the present invention also provides double stranded RNAi agents for inhibiting expression of IGFALS, which comprise a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises at least 15 contiguous nucleotides from nucleotides 11-62, 24-62, 79-117, 79-130, 155-173, 194-216, 194-229, 211-229, 232-293, 254-272, 310-328, 310-349, 324-345, 331-349, 353-371, 353-394, 376-394, 407-425, 439-449, 431-470, 484-515, 497-515, 541-580, 547-568, 596-647, 616-634, 673-691, 694-712, 694-734, 777-799, 781-799, 825-843, 825-855, 869-922, 958-976, 958-988, 1064-1085, 1064-1096, 1067-1085, 1067-1096, 1100-1141, 1111-1129, 1145-1163, 1145-1186, 1159-1186, 1168-1196, 1168-1214, 1193-1214, 1266-1307, 1321-1339, 1342-1373, 1375-1406, 1432-1450, 1454-1472, 1519-1537, 1519-1559, 1534-1555, 1541-1559, 1606-1624, 1606-1637, 1613-1635, 1672-1690, 1672-1712, 1749-1779, 1783-1801, 1805-1823, 1806-1829, 1871-1889, 1871-1919, 1949-1977, 1993-2011, 2013-2042, 2048-2077, 2048-2088, or 2052-2084 of the nucleotide sequence of SEQ ID NO:1, and the antisense strand comprises at least 15 contiguous nucleotides from the corresponding position of the nucleotide sequence of SEQ ID NO: 2 such that the antisense strand is complementary to the at least 15 contiguous nucleotides in the sense strand.

In certain embodiments, the present invention provides double stranded ribonucleic acid (RNAi) agent for inhibiting expression of insulin-like growth factor 1 (IGF-1), which comprise a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises at least 15 contiguous nucleotides selected from the group consisting of nucleotides 330-369, 342-369, 432-490, 432-482, 436-462, 534-559, 330-350, 342-362, 348-368, 349-369, 432-452, 435-455, 436-456, 438-458, 440-460, 441-461, 442-462, 449-469, 455-475, 460-480, 461-481, 462-482, 464-484, 470-490, 484-501, 534-554, 536-556, 538-558, 539-559, 542-562, 548-568, 577-597, 582-602, or 640-660 of the nucleotide sequence of SEQ ID NO: 11 and the antisense strand comprises at least 15 contiguous nucleotides from the corresponding position of the nucleotide sequence of SEQ ID NO: 12 such that the antisense strand is complementary to the at least 15 contiguous nucleotides in the sense strand.

In certain embodiments, the present invention provides double stranded RNAi agents for inhibiting expression of insulin-like growth factor 1 (IGF-1), which comprise a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from nucleotides 6-90, 127-145, 185-238, 247-265, 277-295, 389-417, 430-480, 543-561, 654-690, 750-768, 774-870, 894-930, 1007-1029, 1075-1126, 1144-1162, 1197-1215, 1232-1250, 1293-1311, 1334-1352, 1388-1458, 1463-1490, 1511-1529, 1599-1617, 1643-1661, 1690-1727, 1793-1825, 1843-1861, 2057-2075, 2090-2130, 2192-2228, 2310-2332, 2357-2375, 2521-2539, 2566-2588, 2648-2684, 2793-2811, 2962-2980, 3120-3142, 3208-3233, 3269-3287, 3417-3435, 3449-3467, 3575-3603, 3686-3704, 3721-3739, 3806-3824, 3939-3957, 3982-4018, 4081-4037, 4154-4172, 4271-4289, 4319-4377, 4436-4478, 4484-4502, 4523-4545, 4566-4584, 4610-4660, 4686-4717, 4734-4769, 4780-4798, 4815-4843, 4884-4902, 4911-4929, 5004-5034, 5050-5068, 5171-5256, 5311-5364, 5409-5430, 5551-5588, 5609-5638, 5694-5712, 5715-5758, 5790-5808, 5906-5928, 5934-5952, 6323-6345, 6399-6417, 6461-6497, 6510-6535, 6584-6612, 6629-6647, 6661-6683, 6726-6789, 6796-6824, 6826-6851, 6858-6905, 6910-6927, 7004-7022, 7035-7130, 7144-7162, 7175-7241, and 7252-7270 of the nucleotide sequence of SEQ ID NO: 13, and the antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the corresponding position of the nucleotide sequence of SEQ ID NO: 14 such that the antisense strand is complementary to the at least 15 contiguous nucleotides differing by no more than 3 nucleotides in the sense strand.

In certain embodiments, the agents comprise a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises at least 15 contiguous nucleotides selected from the group of nucleotides 342-369, 432-462, 330-350, 342-362, 348-368, 349-369, 432-452, 435-455, 436-456, 438-458, 440-460, 442-462, 470-490, 481-501, 536-556, or 539-559 of the nucleotide sequence of SEQ ID NO:11 and the antisense strand comprises at least 15 contiguous nucleotides from the corresponding position of the nucleotide sequence of SEQ ID NO: 12 such that the antisense strand is complementary to the at least 15 contiguous nucleotides in the sense strand.

In certain embodiments, the agents comprise a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises at least 15 contiguous nucleotides selected from the group of nucleotides 340-369, 430-490, 430-482, 434-460, 532-559, 328-350, 340-362, 346-368, 347-369, 430-452, 433-455, 434-456, 436-458, 438-460, 439-461, 440-462, 447-469, 453-475, 458-480, 459-481, 460-482, 461-483, 462-484, 468-490, 479-501, 532-554, 534-556, 536-558, 537-559, 540-562, 546-568, 575-597, 580-602, or 638-660 of the nucleotide sequence of SEQ ID NO: 11, for example nucleotides 342-369, 432-462, 330-350, 342-362, 348-368, 349-369, 432-452, 435-455, 436-456, 438-458, 440-460, 442-462, 470-490, 481-501, 536-556, or 539-559 of the nucleotide sequence of SEQ ID NO: 11, and the antisense strand comprises at least 15 contiguous nucleotides from the corresponding position of the nucleotide sequence of SEQ ID NO: 12 such that the antisense strand is complementary to the at least 15 contiguous nucleotides in the sense strand.

In certain embodiments, the agents comprise a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises at least 15 contiguous nucleotides selected from the group of nucleotides 6-90, 127-145, 185-238, 247-265, 277-295, 389-417, 430-480, 543-561, 654-690, 750-768, 774-870, 894-930, 1007-1029, 1075-1126, 1144-1162, 1197-1215, 1232-1250, 1293-1311, 1334-1352, 1388-1458, 1463-1490, 1511-1529, 1599-1617, 1643-1661, 1690-1727, 1793-1825, 1843-1861, 2057-2075, 2090-2130, 2192-2228, 2310-2332, 2357-2375, 2521-2539, 2566-2588, 2648-2684, 2793-2811, 2962-2980, 3120-3142, 3208-3233, 3269-3287, 3417-3435, 3449-3467, 3575-3603, 3686-3704, 3721-3739, 3806-3824, 3939-3957, 3982-4018, 4081-4037, 4154-4172, 4271-4289, 4319-4377, 4436-4478, 4484-4502, 4523-4545, 4566-4584, 4610-4660, 4686-4717, 4734-4769, 4780-4798, 4815-4843, 4884-4902, 4911-4929, 5004-5034, 5050-5068, 5171-5256, 5311-5364, 5409-5430, 5551-5588, 5609-5638, 5694-5712, 5715-5758, 5790-5808, 5906-5928, 5934-5952, 6323-6345, 6399-6417, 6461-6497, 6510-6535, 6584-6612, 6629-6647, 6661-6683, 6726-6789, 6796-6824, 6826-6851, 6858-6905, 6910-6927, 7004-7022, 7035-7130, 7144-7162, 7175-7241, or 7252-7270 of the nucleotide sequence of SEQ ID NO: 13, and the antisense strand comprises at least 15 contiguous nucleotides from the corresponding position of the nucleotide sequence of SEQ ID NO: 14 such that the antisense strand is complementary to the at least 15 contiguous nucleotides in the sense strand.

In certain embodiments, substantially all of the nucleotides of the sense strand are modified nucleotides. In certain embodiments, substantially all of the nucleotides of the antisense strand are modified nucleotides. In certain embodiments, substantially all of the nucleotides of both strands are modified. In preferred embodiments, the double stranded RNAi agent comprises a ligand, e.g., the double stranded RNAi agent is conjugated to at least one ligand, wherein the ligand is one or more GalNAc derivatives attached through a monovalent, a bivalent or a trivalent branched linker.

In certain embodiments, the sense strand and the antisense strand comprise a region of complementarity which comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from any one of the antisense sequences listed in any one of Tables 3, 5, 6, 8, 12, or 14 for IGFALS or any one of Tables 9, 11, 15, 17, 18, or 20 for IGF-1.

For example, in certain embodiments, the sense strand and the antisense strand comprise a region of complementarity which comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from any one of the antisense nucleotide sequences selected from the group of the antisense nucleotide sequence of duplexes targeted to IGF selected from the group AD-66722, AD-66748, AD-66746, AD-66747, AD-66733, AD-66752, AD-66739, AD-66738, AD-66725, AD-66740, AD-66750, AD-66729, AD-66745, AD-66749, AD-66720, AD-66724, AD-66726, AD-66766, AD-66761, AD-66755, AD-66751, AD-66719, AD-66727, AD-66744, AD-66760, AD-66753, AD-66721, AD-66716, AD-66743, or AD-66728-AD-77150, AD-77158, AD-74963, AD-77138, AD-75740, AD-74968, AD-74965, AD-75766, AD-75761, AD-75137, AD-74979, AD-74966, AD-75750, AD-77126, AD-74971, AD-74982, AD-77144, AD-77149, AD-75751, AD-75111, AD-77147, AD-74964, AD-74983, AD-75765, AD-74970, AD-75749, AD-77168, AD-77127, AD-75748, AD-75779, AD-75145, AD-74975, AD-77151, AD-75170, AD-75741, AD-75162, AD-74985, AD-75759, AD-75218, AD-74981, AD-75155, AD-74978, AD-77153, AD-75157, AD-75123, AD-75184, AD-77160, AD-75125, AD-75229, AD-77165, AD-75112, AD-75206, AD-75769, AD-75174, AD-75225, AD-75792, AD-75115, AD-74986, AD-77171, AD-75131, AD-77128, AD-75179, AD-75792, AD-77124, AD-75191, AD-75774, AD-75114, AD-74973, AD-77156, AD-75120, AD-75130, AD-74967, AD-75231, AD-74987, AD-77140, AD-74969, AD-75000, AD-75791, AD-75143, AD-77120, AD-77142, AD-75217, AD-75234, AD-75173, AD-75232, AD-75188, AD-75135, AD-75018, AD-77122, AD-75009, AD-75121, AD-75791, AD-77135, AD-75214, AD-74994, AD-75139, AD-75166, AD-75020, AD-77159, AD-75236, AD-77123, AD-77133, AD-74972, AD-75223, AD-75148, AD-75124, AD-75185, AD-75150, AD-74976, AD-74980, AD-75212, AD-75239, AD-75221, AD-75118, AD-75793, AD-75023, AD-75164, AD-74997, AD-74984, AD-75011, AD-75203, AD-77161, AD-75033, AD-75177, AD-75795, AD-77146, AD-75793, AD-75788, AD-75079, AD-75152, AD-77121, AD-75237, AD-75014, AD-75755, AD-75028, AD-75091, AD-75110, AD-75230, AD-75029, AD-75099, AD-77130, AD-75224, AD-75142, AD-75760, AD-75795, AD-77136, AD-75032, AD-75757, AD-75017, AD-75151, AD-75122, AD-75002, AD-75021, AD-75005, AD-75088, AD-75153, AD-75208, AD-74977, AD-75069, AD-75107, AD-74990, AD-75061, AD-75083, AD-75116, AD-75169, AD-75058, AD-74991, AD-75041, AD-77131, AD-75772, AD-77169, AD-75133, AD-75222, AD-75007, AD-75101, AD-77137, AD-75090, AD-77148, AD-75008, AD-77134, AD-74999, AD-75048, AD-75095, AD-74974, AD-75788, AD-75057, AD-75113, AD-77172, AD-75016, AD-75186, AD-75205, AD-75238, or AD-75146; for example duplexes AD-66722, AD-66748, AD-66746, AD-66747, AD-66733, AD-66752, AD-66739, AD-66738, AD-66725, AD-66740, AD-66750, AD-66729, or AD-66745. In certain embodiments, nucleotide sequences selected from the group duplexes targeted to IGF selected from the group AD-66722, AD-66748, AD-66746, AD-66747, AD-66733, AD-66752, AD-66739, AD-66738, AD-66725, AD-66740, AD-66750, AD-66729, and AD-66745. In certain embodiments, the sense strand and the antisense strand comprise a region of complementarity which comprises at least 15 contiguous nucleotides of any one of the sense and antisense nucleotide sequences of the foregoing duplexes.

In certain embodiments, the sense strand and the antisense strand comprise a region of complementarity which comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from any one of the antisense sequences of the duplexes targeted to IGFALS selected from the group AD-62728, AD-62734, AD-68111, AD-68709, AD-68712, AD-68715, AD-68716, AD-68717, AD-68719, AD-68720, AD-68722, AD-68725, AD-68726, AD-68730, AD-68731, AD-73782, AD-73773, AD-73765, AD-73946, AD-73947, AD-73858, AD-73797, AD-73808, AD-73906, AD-73912, AD-73848, AD-73836, AD-73818, AD-73786, AD-73862, AD-73795, AD-73766, AD-73930, AD-73825, AD-73924, AD-73802, AD-73767, AD-73771, AD-73777, AD-73793, AD-73898, AD-73784, AD-73882, AD-73803, AD-73772, AD-73907, AD-73948, AD-73890, AD-73883, AD-73770, AD-73867, AD-73931, AD-73932, AD-73787, AD-73791, AD-73880, AD-73914, AD-73849, AD-73863, AD-73920, AD-73944, AD-73841, AD-73785, AD-73804, AD-73823, AD-73885, AD-73788, AD-73865, AD-73941, AD-73859, AD-73913, AD-73892, AD-73837, AD-73842, AD-73840, AD-73813, AD-73796, AD-73875, AD-73900, AD-73922, AD-73861, AD-73816, AD-73764, AD-73868, AD-73812, AD-73826, AD-73938, AD-73843, AD-73817, AD-73943, AD-73827, AD-73937, AD-73877, AD-73833, AD-73807, AD-73819, AD-73886, AD-73919, AD-73800, AD-76171, AD-76173, AD-76203, AD-76210, AD-76172, AD-76175, AD-76209, AD-76174, AD-76208, AD-76186, AD-76177, AD-76199, AD-76197, or AD-76212.

In certain embodiments, substantially all of the nucleotides of the sense strand are modified nucleotides. In certain embodiments, substantially all of the nucleotides of the antisense strand are modified nucleotides. In certain embodiments, substantially all of the nucleotides of both strands are modified.

In one embodiment, at least one of the modified nucleotides is selected from the group consisting of a deoxy-nucleotide, a 3′-terminal deoxy-thymine (dT) nucleotide, a 2′-O-methyl modified nucleotide, a 2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an unlocked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2′-amino-modified nucleotide, a 2′-O-allyl-modified nucleotide, 2′-C-alkyl-modified nucleotide, 2′-hydroxly-modified nucleotide, a 2′-methoxyethyl modified nucleotide, a 2′-O-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, a non-natural base comprising nucleotide, a tetrahydropyran modified nucleotide, a 1,5-anhydrohexitol modified nucleotide, a cyclohexenyl modified nucleotide, a nucleotide comprising a phosphorothioate group, a nucleotide comprising a methylphosphonate group, a nucleotide comprising a 5′-phosphate, and a nucleotide comprising a 5′-phosphate mimic. In another embodiment, the modified nucleotides comprise a short sequence of 3′-terminal deoxy-thymine nucleotides (dT).

In certain embodiments, substantially all of the nucleotides of the sense strand are modified. In certain embodiments, substantially all of the nucleotides of the antisense strand are modified. In certain embodiments, substantially all of the nucleotides of both the sense strand and the antisense strand are modified.

In certain embodiments, the duplex comprises a modified antisense nucleotide sequence targeted to IGFALS provided in Table 5, 8, or 14, or targeted to IGF-1 in Table 11, 17, or 20. In certain embodiments, the duplex comprises a modified sense strand nucleotide sequence targeted to IGFALS provided in Table 5, 8, or 14, or targeted to IGF-1 in Table 11, 17, or 20. In certain embodiments, the duplex comprises the modified sense strand nucleotide sequence and the modified antisense strand nucleotide of any one of the duplexes targeted to IGFALS provided in Table 5, 8, or 14, or targeted to IGF-1 in Table 11, 17, or 20.

In certain embodiments, the region of complementarity between the antisense strand and the target is at least 17 nucleotides in length. For example, the region of complementarity between the antisense strand and the target is 19 to 21 nucleotides in length, for example, the region of complementarity is 21 nucleotides in length. In preferred embodiments, each strand is no more than 30 nucleotides in length.

In some embodiments, at least one strand comprises a 3′ overhang of at least 1 nucleotide, e.g., at least one strand comprises a 3′ overhang of at least 2 nucleotides, e.g., 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In other embodiments, at least one strand of the RNAi agent comprises a 5′ overhang of at least 1 nucleotide. In certain embodiments, at least one strand comprises a 5′ overhang of at least 2 nucleotides, e.g., 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In still other embodiments, both the 3′ and the 5′ end of one strand of the RNAi agent comprise an overhang of at least 1 nucleotide. In other embodiment, at least one strand comprises a 3′ overhang of at least 2 nucleotides.

In many embodiments, the double stranded RNAi agent further comprises a ligand. The ligand may be one or more GalNAc attached to the RNAi agent through a monovalent, a bivalent, or a trivalent branched linker. The ligand may be conjugated to the 3′ end of the sense strand of the double stranded RNAi agent. The ligand can be an N-acetylgalactosamine (GalNAc) derivative including, but not limited to

In various embodiments, the ligand is attached to the 5′ end of the sense strand of the double stranded RNAi agent, the 3′ end of the antisense strand of the double stranded RNAi agent, or the 5′ end of the antisense strand of the double stranded RNAi agent.

In some embodiments, the double stranded RNAi agents of the invention comprise a plurality, e.g., 2, 3, 4, 5, or 6, of GalNAc, each independently attached to a plurality of nucleotides of the double stranded RNAi agent through a plurality of monovalent linkers.

In certain embodiments, the dsRNAi is agent conjugated to the ligand as shown in the following schematic:

and, wherein X is O or S. In one embodiment, the X is O.

In certain embodiments, the ligand is a cholesterol.

In certain embodiments, the region of complementarity comprises any one of the antisense sequences targeted to IGFALS provided in Table 3, 5, 6, 8, 12, or 14 or targeted to IGF-1 in Table 9, 11, 15, 17, 18, or 20. In another embodiment, the region of complementarity consists of any one of the antisense sequences of targeted to IGFALS provided in Table 3, 5, 6, 8, 12, or 14 or targeted to IGF-1 in Table 9, 11, 15, 17, 18, or 20.

In another aspect, the invention provides a double stranded RNAi agent for inhibiting expression of IGFALS or IGF-1, wherein the double stranded RNAi agent comprises a sense strand complementary to an antisense strand, wherein the antisense strand comprises a region complementary to part of an mRNA encoding IGFALS or IGF-1, wherein each strand is about 14 to about 30 nucleotides in length, wherein the double stranded RNAi agent is represented by formula (III):

(III) sense: 5′ n_(p)-N_(a)-(X X X)_(i)-N_(b)-YYY-N_(b)-(Z Z Z)_(j)-N_(a)-n_(q) 3′ antisense: 3′ n_(p′)-N_(a′)-(X′X′X′)_(k)-N_(b′)-Y′Y′Y′-N_(b′)-(Z′Z′Z′)_(l)- N_(p′)-n_(q′) 5′

wherein: i, j, k, and 1 are each independently 0 or 1; p, p′, q, and q′ are each independently 0-6; each N_(a) and N_(a)′ independently represents an oligonucleotide sequence comprising 0-25 nucleotides which are either modified or unmodified or combinations thereof, each sequence comprising at least two differently modified nucleotides; each N_(b) and N_(b)′ independently represents an oligonucleotide sequence comprising 0-10 nucleotides which are either modified or unmodified or combinations thereof; each n_(p), n_(p)′, n_(q), and n_(q)′, each of which may or may not be present, independently represents an overhang nucleotide; XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently represent one motif of three identical modifications on three consecutive nucleotides; modifications on N_(b) differ from the modification on Y and modifications on N_(b)′ differ from the modification on Y; and wherein the double stranded RNAi agent comprises a ligand, e.g., the sense strand is conjugated to at least one ligand.

In certain embodiments, i is 0; j is 0; i is 1; j is 1; both i and j are 0; or both i and j are 1. In another embodiment, k is 0; l is 0; k is 1; l is 1; both k and l are 0; or both k and l are 1. In another embodiment, XXX is complementary to X′X′X′, YYY is complementary to Y′Y′Y′, and ZZZ is complementary to Z′Z′Z′. In another embodiment, the YYY motif occurs at or near the cleavage site of the sense strand. In another embodiment, the Y′Y′Y′ motif occurs at the 11, 12 and 13 positions of the antisense strand from the 5′-end. In one embodiment, the Y′ is 2′-O-methyl.

For example, formula (III) can be represented by formula (Ma):

(IIIa) sense: 5′ n_(p)-N_(a)-Y Y Y-N_(a)-n_(q) 3′ antisense: 3′ n_(p′)-N_(a′)-Y′Y′Y′-N_(a′)-n_(q′) 5′.

In another embodiment, formula (III) is represented by formula (IIIb):

(IIIb) sense: 5′ n_(p)-N_(a)-Y Y Y-N_(b)-Z Z Z-N_(a)-n_(q) 3′ antisense: 3′ n_(p)-N_(a)-Y′Y′Y′-N_(b)-Z′Z′Z′-N_(a′)-n_(q′) 5′

wherein each N_(b) and N_(b)′ independently represents an oligonucleotide sequence comprising 1-5 modified nucleotides.

Alternatively, formula (III) can be represented by formula (IIIc):

sense: 5′ n_(p) -N_(a) -X X X -N_(b) -Y Y Y -N_(a) - n_(q) 3′ antisense: 3′ n_(p′)-N_(a′)- X′X′X′-N_(b′)- Y′Y′Y′- N_(a′)- n_(q′) 5′ (IIIc)

wherein each N_(b) and N_(b)′ independently represents an oligonucleotide sequence comprising 1-5 modified nucleotides.

Further, formula (III) can be represented by formula (IIId):

sense: 5′ n_(p) -N_(a) -X X X- N_(b) -Y Y Y -N_(b) -Z Z Z -N_(a) - n_(q) 3′ antisense: 3′ n_(p′)-N_(a′)- X′X′X′- N_(b′)-Y′Y′Y′-N_(b′)-Z′Z′Z′- N_(a′)- n_(q′) 5′ (IIId)

wherein each N_(b) and N_(b)′ independently represents an oligonucleotide sequence comprising 1-5 modified nucleotides and each N_(a) and N_(a)′ independently represents an oligonucleotide sequence comprising 2-10 modified nucleotides.

In certain embodiment, the double stranded region is 15-30 nucleotide pairs in length. For example, the double stranded region can be 17-23 nucleotide pairs in length. The double stranded region can be 17-25 nucleotide pairs in length. The double stranded region can be 23-27 nucleotide pairs in length. The double stranded region can be 19-21 nucleotide pairs in length. The double stranded region can be 21-23 nucleotide pairs in length.

In certain embodiments, each strand has 15-30 nucleotides. In other embodiments, each strand has 19-30 nucleotides.

Modifications on the nucleotides are selected from the group including, but not limited to, LNA, HNA, CeNA, 2′-methoxyethyl, 2′-O-alkyl, 2′-O-allyl, 2′-C-allyl, 2′-fluoro, 2′-deoxy, 2′-hydroxyl, and combinations thereof. In another embodiment, the modifications on the nucleotides are 2′-O-methyl or 2′-fluoro modifications.

In many embodiments, the double stranded RNAi agent further comprises a ligand. The ligand may be one or more GalNAc attached to the RNAi agent through a monovalent, a bivalent, or a trivalent branched linker. The ligand may be conjugated to the 3′ end of the sense strand of the double stranded RNAi agent. The ligand can be an N-acetylgalactosamine (GalNAc) derivative including, but not limited to

In various embodiments, the ligand is attached to the 5′ end of the sense strand of the double stranded RNAi agent, the 3′ end of the antisense strand of the double stranded RNAi agent, or the 5′ end of the antisense strand of the double stranded RNAi agent.

In some embodiments, the double stranded RNAi agents of the invention comprise a plurality, e.g., 2, 3, 4, 5, or 6, of GalNAc, each independently attached to a plurality of nucleotides of the double stranded RNAi agent through a plurality of monovalent linkers.

An exemplary structure of a dsRNAi agent conjugated to the ligand is shown in the following schematic

In certain embodiments, the ligand can be a cholesterol.

In certain embodiments, the double stranded RNAi agent further comprises at least one phosphorothioate or methylphosphonate internucleotide linkage. For example the phosphorothioate or methylphosphonate internucleotide linkage can be at the 3′-terminus of one strand, i.e., the sense strand or the antisense strand; or at the ends of both strands, the sense strand and the antisense strand. In certain embodiments, the phosphorothioate or methylphosphonate internucleotide linkage is at the 5′-terminus of one strand, i.e., the sense strand or the antisense strand; or at the ends of both strands, the sense strand and the antisense strand.

In certain embodiments, the phosphorothioate or methylphosphonate internucleotide linkage is at the both the 5′- and 3′-terminus of one strand, i.e., the sense strand or the antisense strand; or at the ends of both strands, the sense strand and the antisense strand.

In certain embodiments, the base pair at the 1 position of the 5′-end of the antisense strand of the duplex is an AU base pair.

In certain embodiments, the Y nucleotides contain a 2′-fluoro modification. In another embodiment, the Y′ nucleotides contain a 2′-O-methyl modification. In another embodiment, p′>0. In some embodiments, p′=2. In some embodiments, q′=0, p=0, q=0, and p′ overhang nucleotides are complementary to the target mRNA. In some embodiments, q′=0, p=0, q=0, and p′ overhang nucleotides are non-complementary to the target mRNA.

In certain embodiments, the sense strand has a total of 21 nucleotides and the antisense strand has a total of 23 nucleotides.

In certain embodiments, at least one n_(p)′ is linked to a neighboring nucleotide via a phosphorothioate linkage. In other embodiments, all n_(p)′ are linked to neighboring nucleotides via phosphorothioate linkages.

In certain embodiments, the dsRNAi agent is selected from the group of any one of the double stranded RNAi agents targeted to IGFALS provided in Table 3, 5, 6, 8, 12, or 14, or targeted to IGF-1 in Table 9, 11, 15, 17, 18, or 20. In certain embodiments, all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand comprise a modification.

In an aspect, the invention provides a double stranded RNAi agent for inhibiting expression of IGFALS or IGF-1 in a cell, wherein the double stranded RNAi agent comprises a sense strand complementary to an antisense strand, wherein the antisense strand comprises a region complementary to part of an mRNA encoding IGFALS or IGF-1, wherein each strand is about 14 to about 30 nucleotides in length, wherein the double stranded RNAi agent is represented by formula (III):

sense: 5′ n_(p) -N_(a) -(X X X) _(i)-N_(b) -Y Y Y -N_(b) -(Z Z Z)_(j)-N_(a) - n_(q) 3′ antisense: 3′ n_(p)′-N_(a)′-(X′X′X′)_(k) -N_(b)′-Y′Y′Y′-N_(b)′-(Z′Z′Z′)_(l)-N_(a)′- n_(q)′ 5′ (III)

wherein i, j, k, and 1 are each independently 0 or 1; p, p′, q, and q′ are each independently 0-6; each N_(a) and N_(a)′ independently represents an oligonucleotide sequence comprising 0-25 nucleotides which are either modified or unmodified or combinations thereof, each sequence comprising at least two differently modified nucleotides; each N_(b) and N_(b)′ independently represents an oligonucleotide sequence comprising 0-10 nucleotides which are either modified or unmodified or combinations thereof; each n_(p), n_(p)′, n_(q), and n_(q)′, each of which may or may not be present independently represents an overhang nucleotide; XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently represent one motif of three identical modifications on three consecutive nucleotides, and wherein the modifications are 2′-O-methyl or 2′-fluoro modifications; modifications on N_(b) differ from the modification on Y and modifications on N_(b)′ differ from the modification on Y′; and wherein the double stranded RNAi agent comprises a ligand, e.g., the double stranded RNAi agent is conjugated to at least one ligand, wherein the ligand is one or more GalNAc derivatives attached through a monovalent, a bivalent or a trivalent branched linker.

In an aspect, the invention provides a double stranded RNAi agent for inhibiting expression of IGFALS or IGF-1 in a cell, wherein the double stranded RNAi agent comprises a sense strand complementary to an antisense strand, wherein the antisense strand comprises a region complementary to part of an mRNA encoding IGFALS or IGF-1, wherein each strand is about 14 to about 30 nucleotides in length, wherein the double stranded RNAi agent is represented by formula (III):

sense: 5′ n_(p) -N_(a) -(X X X) _(i)-N_(b) -Y Y Y -N_(b) -(Z Z Z)_(j)-N_(a) - n_(q) 3′ antisense: 3′ n_(p)′-N_(a)′-(X′X′X′)_(k)-N_(b)′-Y′Y′Y′-N_(b)′-(Z′Z′Z′)_(l)-N_(a)′- n_(q)′ 5′ (III)

wherein: i, j, k, and 1 are each independently 0 or 1; each n_(p), n_(q), and n_(q)′, each of which may or may not be present, independently represents an overhang nucleotide;

p, q, and q′ are each independently 0-6; n_(p)′>0 and at least one n_(p)′ is linked to a neighboring nucleotide via a phosphorothioate linkage; each N_(a) and N_(a)′ independently represents an oligonucleotide sequence comprising 0-25 nucleotides which are either modified or unmodified or combinations thereof, each sequence comprising at least two differently modified nucleotides; each N_(b) and N_(b)′ independently represents an oligonucleotide sequence comprising 0-10 nucleotides which are either modified or unmodified or combinations thereof; XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently represent one motif of three identical modifications on three consecutive nucleotides, and wherein the modifications are 2′-O-methyl or 2′-fluoro modifications; modifications on N_(b) differ from the modification on Y and modifications on N_(b)′ differ from the modification on Y; and wherein the double stranded RNAi agent comprises a ligand, e.g., the double stranded RNAi agent is conjugated to at least one ligand, wherein the ligand is one or more GalNAc derivatives attached through a monovalent, a bivalent or a trivalent branched linker.

In certain embodiments, the invention provides a d double stranded ribonucleic acid (RNAi) agent for inhibiting expression of IGFALS or IGF-1, wherein the double stranded RNAi agent comprises a sense strand complementary to an antisense strand, wherein the antisense strand comprises a region complementary to part of an mRNA encoding IGFALS or IGF-1, wherein each strand is about 14 to about 30 nucleotides in length, wherein the double stranded RNAi agent is represented by formula (III):

sense: 5′ n_(p)-N_(a)-(X X X) _(i)-N_(b) -Y Y Y -N_(b) -(Z Z Z)_(j)-N_(a) - n_(q) 3′ antisense: 3′ n_(p)′-N_(a)′-(X′X′X′)_(k)-N_(b)′-Y′Y′Y′-N_(b)′-(Z′Z′Z′)_(l)-N_(a)′- n_(q)′ 5′ (III)

wherein i, j, k, and 1 are each independently 0 or 1; each n_(p), n_(q), and n_(q)′, each of which may or may not be present, independently represents an overhang nucleotide; p, q, and q′ are each independently 0-6; n_(p)′>0 and at least one n_(p)′ is linked to a neighboring nucleotide via a phosphorothioate linkage; each N_(a) and N_(a)′ independently represents an oligonucleotide sequence comprising 0-25 nucleotides which are either modified or unmodified or combinations thereof, each sequence comprising at least two differently modified nucleotides; each N_(b) and N_(b)′ independently represents an oligonucleotide sequence comprising 0-10 nucleotides which are either modified or unmodified or combinations thereof; XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently represent one motif of three identical modifications on three consecutive nucleotides, and wherein the modifications are 2′-O-methyl or 2′-fluoro modifications; modifications on N_(b) differ from the modification on Y and modifications on N_(b)′ differ from the modification on Y; and wherein the double stranded RNAi agent is conjugated to at least one ligand, wherein the ligand is one or more GalNAc derivatives attached through a monovalent, a bivalent or a trivalent linker.

In an aspect, the invention provides a double stranded RNAi agent for inhibiting expression of IGFALS or IGF-1, wherein the double stranded RNAi agent comprises a sense strand complementary to an antisense strand, wherein the antisense strand comprises a region complementary to part of an mRNA encoding IGFALS or IGF-1, wherein each strand is about 14 to about 30 nucleotides in length, wherein the double stranded RNAi agent is represented by formula

sense: 5′ n_(p) -N_(a) -(X X X) _(i)-N_(b) -Y Y Y -N_(b) -(Z Z Z)_(j)-N_(a) - n_(q) 3′ antisense: 3′ n_(p)′-N_(a)′-(X′X′X′)_(k)-N_(b)′-Y′Y′Y′-N_(b)′-(Z′Z′Z′)_(l)-N_(a)′- n_(q)′ 5′ (III)

wherein i, j, k, and 1 are each independently 0 or 1; each n_(p), n_(q), and n_(q)′, each of which may or may not be present, independently represents an overhang nucleotide; p, q, and q′ are each independently 0-6; n_(p)′>0 and at least one n_(p)′ is linked to a neighboring nucleotide via a phosphorothioate linkage; each N_(a) and N_(a)′ independently represents an oligonucleotide sequence comprising 0-25 nucleotides which are either modified or unmodified or combinations thereof, each sequence comprising at least two differently modified nucleotides; each N_(b) and N_(b)′ independently represents an oligonucleotide sequence comprising 0-10 nucleotides which are either modified or unmodified or combinations thereof; XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently represent one motif of three identical modifications on three consecutive nucleotides, and wherein the modifications are 2′-O-methyl or 2′-fluoro modifications; modifications on N_(b) differ from the modification on Y and modifications on N_(b)′ differ from the modification on Y; wherein the double stranded RNAi agent comprises a ligand, e.g., the double stranded RNAi agent is conjugated to at least one ligand, wherein the ligand is one or more GalNAc derivatives attached through a monovalent, a bivalent or a trivalent branched linker.

In an aspect, the invention provides a double stranded RNAi agent capable of inhibiting the expression of IGFALS or IGF-1 in a cell, wherein the double stranded RNAi agent comprises a sense strand complementary to an antisense strand, wherein the antisense strand comprises a region complementary to part of an mRNA encoding IGFALS of IGF-1, wherein each strand is about 14 to about 30 nucleotides in length, wherein the double stranded RNAi agent is represented by formula (III):

sense: 5′ n_(p) -N_(a) -(X X X) _(i)-N_(b) -Y Y Y -N_(b) -(Z Z Z)_(j)-N_(a) - n_(q) 3′ antisense: 3′ n_(p)′-N_(a)′-(X′X′X′)_(k)-N_(b)′-Y′Y′Y′-N_(b)′-(Z′Z′Z′)_(l)-N_(a)′- n_(q)′ 5′ (III)

wherein i, j, k, and 1 are each independently 0 or 1; each n_(p), n_(q), and n_(q)′, each of which may or may not be present, independently represents an overhang nucleotide; p, q, and q′ are each independently 0-6; n_(p)′>0 and at least one n_(p)′ is linked to a neighboring nucleotide via a phosphorothioate linkage; each N_(a) and N_(a)′ independently represents an oligonucleotide sequence comprising 0-25 nucleotides which are either modified or unmodified or combinations thereof, each sequence comprising at least two differently modified nucleotides; each N_(b) and N_(b)′ independently represents an oligonucleotide sequence comprising 0-10 nucleotides which are either modified or unmodified or combinations thereof; XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently represent one motif of three identical modifications on three consecutive nucleotides, and wherein the modifications are 2′-O-methyl or 2′-fluoro modifications; modifications on N_(b) differ from the modification on Y and modifications on N_(b)′ differ from the modification on Y; wherein the sense strand comprises at least one phosphorothioate linkage; and wherein the double stranded RNAi agent comprises a ligand, e.g., the double stranded RNAi agent is conjugated to at least one ligand, wherein the ligand is one or more GalNAc derivatives attached through a monovalent, a bivalent or a trivalent branched linker.

In an aspect, the invention provides a double stranded RNAi agent for inhibiting expression of IGFALS or IGF-1 in a cell, wherein the double stranded RNAi agent comprises a sense strand complementary to an antisense strand, wherein the antisense strand comprises a region complementary to part of an mRNA encoding IGFALS or IGF-1, wherein each strand is about 14 to about 30 nucleotides in length, wherein the double stranded RNAi agent is represented by formula (III):

sense: 5′ n_(p) -N_(a) -Y Y Y - N_(a) - n_(q) 3′ antisense: 3′ n_(p)′-N_(a)′- Y′Y′Y′- N_(a)′- n_(q)′ 5′ (IIIa)

wherein each n_(p), n_(q), and n_(q)′, each of which may or may not be present, independently represents an overhang nucleotide; p, q, and q′ are each independently 0-6; n_(p)′>0 and at least one n_(p)′ is linked to a neighboring nucleotide via a phosphorothioate linkage; each N_(a) and N_(a)′ independently represents an oligonucleotide sequence comprising 0-25 nucleotides which are either modified or unmodified or combinations thereof, each sequence comprising at least two differently modified nucleotides; YYY and Y′Y′Y′ each independently represent one motif of three identical modifications on three consecutive nucleotides, and wherein the modifications are 2′-O-methyl or 2′-fluoro modifications; wherein the sense strand comprises at least one phosphorothioate linkage; wherein the double stranded RNAi agent comprises a ligand, e.g., the double stranded RNAi agent is conjugated to at least one ligand, wherein the ligand is one or more GalNAc derivatives attached through a monovalent, a bivalent or a trivalent branched linker.

In an aspect, the invention provides a double stranded ribonucleic acid (RNAi) agent for inhibiting expression of IGFALS, wherein the double stranded RNAi agent comprises a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:1 and the antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:2, wherein substantially all of the nucleotides of the sense strand comprise a modification selected from a 2′-O-methyl modification and a 2′-fluoro modification, wherein the sense strand comprises two phosphorothioate internucleotide linkages at the 5′-terminus, wherein substantially all of the nucleotides of the antisense strand comprise a modification selected from a 2′-O-methyl modification and a 2′-fluoro modification, wherein the antisense strand comprises two phosphorothioate internucleotide linkages at the 5′-terminus and two phosphorothioate internucleotide linkages at the 3′-terminus, and wherein the sense strand is conjugated to one or more GalNAc derivatives attached through a monovalent, a bivalent or a trivalent linker at the 3′-terminus.

In an aspect, the invention provides a double stranded ribonucleic acid (RNAi) agent for inhibiting expression of insulin-like growth factor 1 (IGF-1), wherein the double stranded RNAi agent comprises a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:11 or 13 and the antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:12 or 14, wherein substantially all of the nucleotides of the sense strand comprise a modification selected from a 2′-O-methyl modification and a 2′-fluoro modification, wherein the sense strand comprises two phosphorothioate internucleotide linkages at the 5′-terminus, wherein substantially all of the nucleotides of the antisense strand comprise a modification selected from a 2′-O-methyl modification and a 2′-fluoro modification, wherein the antisense strand comprises two phosphorothioate internucleotide linkages at the 5′-terminus and two phosphorothioate internucleotide linkages at the 3′-terminus, and wherein the sense strand is conjugated to one or more GalNAc derivatives attached through a monovalent, a bivalent or a trivalent linker at the 3′-terminus.

In certain embodiments, all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand are modified nucleotides. In certain embodiments, each strand has 19-30 nucleotides.

In certain embodiments, substantially all of the nucleotides of the sense strand are modified. In certain embodiments, substantially all of the nucleotides of the antisense strand are modified. In certain embodiments, substantially all of the nucleotides of both the sense strand and the antisense strand are modified.

In an aspect, the invention provides a cell containing the dsRNAi agent as described herein.

In an aspect, the invention provides a vector encoding at least one strand of a dsRNAi agent, wherein the RNAi agent comprises a region of complementarity to at least a part of an mRNA encoding IGFALS or IGF-1, wherein the RNAi is 30 base pairs or less in length, and wherein the RNAi agent targets the mRNA for cleavage. In certain embodiments, the region of complementarity is at least 15 nucleotides in length. In certain embodiments, the region of complementarity is 19 to 23 nucleotides in length.

In an aspect, the invention provides a cell comprising a vector as described herein.

In an aspect, the invention provides a pharmaceutical composition for inhibiting expression of an IGFALS or IGF-1 gene, comprising a double stranded RNAi agent of the invention. In one embodiment, the RNAi agent is administered in an unbuffered solution. In certain embodiments, the unbuffered solution is saline or water. In other embodiments, the RNAi agent is administered with a buffer solution. In such embodiments, the buffer solution can comprise acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof. For example, the buffer solution can be phosphate buffered saline (PBS).

In an aspect, the invention provides a pharmaceutical composition comprising the double stranded RNAi agent of the invention and a lipid formulation. In certain embodiments, the lipid formulation comprises a LNP. In certain embodiments, the lipid formulation comprises MC3.

In an aspect, the invention provides a method of inhibiting IGFALS or IGF-1 expression in a cell, the method comprising (a) contacting the cell with the double stranded RNAi agent of the invention or a pharmaceutical composition of the invention; and (b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of an IGFALS or IGF-1 gene, thereby inhibiting expression of the IGFALS or IGF-1 gene in the cell. In certain embodiments, the cell is within a subject, for example, a human subject, for example a female human or a male human. In preferred embodiments, IGFALS or IGF-1 expression is inhibited by at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%, or to below the threshold of detection of the assay method used. Preferably the expression is inhibited by at least 50%. In some embodiments of the methods of the invention, expression of an IGF-1 gene is inhibited by at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the difference between the elevated level associated with the disease and a normal level in an appropriate control subject. Preferably the elevated level is inhibited by at least 50%.

In an aspect, the invention provides a method of treating a subject having a disease or disorder that would benefit from reduction in IGFALS or IGF-1 expression, such as an IGF system-associated disease or disorder, the method comprising administering to the subject a therapeutically effective amount of a double stranded RNAi agent of the invention or a pharmaceutical composition of the invention, thereby treating the subject.

In an aspect, the invention provides a method of preventing at least one symptom in a subject having a disease or disorder that would benefit from reduction in IGFALS or IGF-1 expression, such as an IGF system-associated disease or disorder, the method comprising administering to the subject a prophylactically effective amount of a double stranded RNAi agent of the invention or a pharmaceutical composition of the invention, thereby preventing at least one symptom in the subject having a disorder that would benefit from reduction in IGFALS or IGF-1 expression.

In certain embodiments, the administration of the double stranded RNAi to the subject causes a decrease in the IGF-1 signaling pathway. In certain embodiments, the administration of the double stranded RNAi causes a decrease in the level of IGF-1 or IGFALS in the subject, e.g., serum levels of IGF-1 or IGFALS in the subject.

In certain embodiments, the IGF system-associated disease is acromegaly. In certain embodiments, the IGF system-associated disease is gigantism. In another embodiment, the IGF system-associated disease is cancer. In certain embodiments, the cancer is metastatic cancer.

In certain embodiments, the invention further comprises administering an inhibitor of growth hormone to a subject with an IGF system-associated disease.

In certain embodiments, the invention further comprises administering an inhibitor of the IGF pathway signaling to a subject with an IGF system-associated disease.

In certain embodiments, wherein the IGF system-associated disease is acromegaly or gigantism, the subject is further treated for acromegaly or gigantism. In certain embodiments, the treatment for acromegaly or gigantism includes surgery. In certain embodiments, the treatment for acromegaly or gigantism includes radiation. In certain embodiments, the treatment for acromegaly or gigantism includes administration of a therapeutic agent.

In certain embodiments, wherein the IGF system-associated disease is cancer, the subject is further treated for cancer. In certain embodiments, the treatment for cancer includes surgery. In certain embodiments, the treatment for cancer includes radiation. In certain embodiments, the treatment for cancer includes administration of a chemotherapeutic agent.

In various embodiments, the dsRNAi agent is administered at a dose of about 0.01 mg/kg to about 10 mg/kg or about 0.5 mg/kg to about 50 mg/kg. In some embodiments, the dsRNAi agent is administered at a dose of about 10 mg/kg to about 30 mg/kg. In certain embodiments, the dsRNAi agent is administered at a dose selected from 0.5 mg/kg 1 mg/kg, 1.5 mg/kg, 3 mg/kg, 5 mg/kg, 10 mg/kg, and 30 mg/kg. In certain embodiments, the dsRNAi agent is administered about once per week, once per month, once every other two months, or once a quarter (i.e., once every three months) at a dose of about 0.1 mg/kg to about 5.0 mg/kg.

In certain embodiments, the double stranded RNAi agent is administered to the subject once a week. In certain embodiments, the dsRNAi agent is administered to the subject once a month. In certain embodiments, the dsRNAi agent is administered once per quarter (i.e., every three months).

In some embodiment, the dsRNAi agent is administered to the subject subcutaneously.

In various embodiments, the methods of the invention further comprise determining the level of IGF-1 in the subject. In certain embodiments, a decrease in the level of expression or activity of the IGF-1 signaling pathway indicates that the IGF system-associated disease is being treated.

In various embodiments, a surrogate marker of IGF-1 expression is measured. In certain embodiments, a change, preferably a clinically relevant change in the surrogate marker indicating effective treatment of diseases associated with an elevated IGF-level are detected, e.g., decreased serum IGF. In the treatment of acromegaly, a clinically relevant change in one or more signs or symptoms associated with acromegaly as provided below can be used as a surrogate marker for a reduction in IGF-1 expression. In the treatment of cancer, a demonstration of stabilization or reduction of tumor burden using RECIST criteria can be used as a surrogate marker for a reduction of IGF-1 expression or activity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic showing various aspects of the IGF-1 signaling pathways.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides iRNA compositions which effect the RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of an Insulin-like Growth Factor Binding Protein, Acid Labile Subunit (IGFALS) or Insulin-like Growth Factor 1 (IGF-1) gene. The gene may be within a cell, e.g., a cell within a subject, such as a human. The use of these iRNAs enables the targeted degradation of mRNAs of the corresponding gene (IGFALS or IGF-1 gene) in mammals.

The iRNAs of the invention have been designed to target a human IGFALS or a human IGF-1 gene, including portions of the gene that are conserved in the IGFALS or IGF-1 othologs of other mammalian species. Without intending to be limited by theory, it is believed that a combination or sub-combination of the foregoing properties and the specific target sites or the specific modifications in these iRNAs confer to the iRNAs of the invention improved efficacy, stability, potency, durability, and safety.

Accordingly, the present invention also provides methods for treating a subject having a disorder that would benefit from inhibiting or reducing the expression of an IGFALS or IGF-1 gene, e.g., an IGF system-associated disease, such as acromegaly or cancer, such as a cancer in which the tumor expresses IGF-1, using iRNA compositions which effect the RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of an IGFALS or an IGF-1 gene.

Very low dosages of the iRNAs of the invention, in particular, can specifically and efficiently mediate RNA interference (RNAi), resulting in significant inhibition of expression of the corresponding target gene (IGFALS or IGF-1 gene).

The iRNAs of the invention include an RNA strand (the antisense strand) having a region which is about 30 nucleotides or less in length, e.g., 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length, which region is substantially complementary to at least part of an mRNA transcript of an IGFALS or IGF-1 gene.

In certain embodiments, the iRNAs of the invention include an RNA strand (the antisense strand) which can include longer lengths, for example up to 66 nucleotides, e.g., 36-66, 26-36, 25-36, 31-60, 22-43, 27-53 nucleotides in length with a region of at least 19 contiguous nucleotides that is substantially complementary to at least a part of an mRNA transcript of an IGFALS or an IGF-1 gene.

In some embodiments, the iRNA agents for use in the methods of the invention include an RNA strand (the antisense strand) which can be up to 66 nucleotides in length, e.g., 36-66, 26-36, 25-36, 31-60, 22-43, 27-53 nucleotides in length, with a region of at least 19 contiguous nucleotides that is substantially complementary to at least a part of an mRNA transcript of an IGFALS or an IGF-1 gene. In some embodiments, such iRNA agents having longer length antisense strands preferably include a second RNA strand (the sense strand) of 20-60 nucleotides in length wherein the sense and antisense strands form a duplex of 18-30 contiguous nucleotides.

Using in vitro and in vivo assays, the present inventors have demonstrated that iRNAs targeting an IGFALS gene or an or IGF-1 gene can mediate RNAi, resulting in significant inhibition of expression of IGFALS or IGF-1, as well as reducing signaling through the IGF-1 pathway which will decrease one or more of the symptoms associated with an IGF system-associated disease, such as acromegaly or cancer. Thus, methods and compositions including these iRNAs are useful for treating a subject having an IGF system-associated disease, such as acromegaly or cancer. The methods and compostions herein are useful for reducing the level of IGFALS or IGF-1 in a subject, e.g., serum or liver IGF-1 in a subject, especially in a subject with acromegaly or a tumor, such as an IGF-1 expressing tumor.

The following detailed description discloses how to make and use compositions containing iRNAs to inhibit the expression of an IGFALS gene or an IGF gene as well as compositions, uses, and methods for treating subjects having diseases and disorders that would benefit from reduction of the expression of an IGFALS gene or an IGF gene.

I. Definitions

In order that the present invention may be more readily understood, certain terms are first defined. In addition, it should be noted that whenever a value or range of values of a parameter are recited, it is intended that values and ranges intermediate to the recited values are also intended to be part of this invention.

The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element, e.g., a plurality of elements.

The term “including” is used herein to mean, and is used interchangeably with, the phrase “including but not limited to”.

The term “or” is used herein to mean, and is used interchangeably with, the term “and/or,” unless context clearly indicates otherwise. For example, “sense strand or antisense strand” is understood as “sense strand or antisense strand or sense strand and antisense strand.”

The term “about” is used herein to mean within the typical ranges of tolerances in the art. For example, “about” can be understood as about 2 standard deviations from the mean. In certain embodiments, about means±10%. In certain embodiments, about means±5%. When about is present before a series of numbers or a range, it is understood that “about” can modify each of the numbers in the series or range.

The term “at least” prior to a number or series of numbers is understood to include the number adjacent to the term “at least”, and all subsequent numbers or integers that could logically be included, as clear from context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, “at least 18 nucleotides of a 21 nucleotide nucleic acid molecule” means that 18, 19, 20, or 21 nucleotides have the indicated property. When at least is present before a series of numbers or a range, it is understood that “at least” can modify each of the numbers in the series or range.

As used herein, “no more than” or “less than” is understood as the value adjacent to the phrase and logical lower values or intergers, as logical from context, to zero. For example, a duplex with an overhang of “no more than 2 nucleotides” has a 2, 1, or 0 nucleotide overhang. When “no more than” is present before a series of numbers or a range, it is understood that “no more than” can modify each of the numbers in the series or range.

As used herein, ranges include both the upper and lower limit.

In the event of a conflict between a sequence and its indicated site on a transcript or other sequence, the nucleotide sequence recited in the specification takes precedence.

Various embodiments of the invention can be combined as determined appropriate by one of skill in the art.

As used herein, “insulin-like growth factor binding protein, acid labile subunit” or “IGFALS” is a serum protein that binds insulin-like growth factors, increasing their half-life and their vascular localization. Production of the encoded protein, predominantly in the liver, which contains twenty leucine-rich repeats, is stimulated by growth hormone. Defects in this gene are a cause of acid-labile subunit deficiency, which manifests itself in delayed and slow puberty. Three transcript variants encoding two different isoforms have been found for this gene. The gene can also be known as ALS or ACLSD. Further information on IGFALS is provided, for example in the NCBI Gene database at www.ncbi.nlm.nih.gov/gene/3483 (which is incorporated herein by reference as of the date of filing this application).

As used herein, “insulin-like growth factor binding protein, acid labile subunit,” used interchangeably with the term “IGFALS,” refers to the naturally occurring gene that encodes an IGF-1 binding protein. The amino acid and complete coding sequences of the reference sequence of the human IGFALS gene may be found in, for example, GenBank Accession No. GI: 225579150 (RefSeq Accession No. NM 004970.2; SEQ ID NO:1; SEQ ID NO:2), GenBank Accession No. GI:225579151 (RefSeq Accession No. NM_001146006.1; SEQ ID NO: 9 and 10). Mammalian orthologs of the human IGFALS gene may be found in, for example, GI:142388344 (RefSeq Accession No. NM 008340.3, mouse; SEQ ID NO:3 and SEQ ID NO:4); GI:71896591 (RefSeq Accession No. NM 053329.2, rat; SEQ ID NO:5 and SEQ ID NO:6); GenBank Accession Nos. GI:544514850 (RefSeq Accession No. XM_005590898.1, cynomolgus monkey; SEQ ID NO:7 and SEQ ID NO:8).

A number of naturally occurring SNPs are known and can be found, for example, in the SNP database at the NCBI at www.ncbi.nlm.nih.gov/SNP/snp_ref.cgi?locusld=3483 (which is incorporated herein by reference as of the date of filing this application) which lists SNPs in human IGFALS. In preferred embodiments, such naturally occurring variants are included within the scope of the IGFALS gene sequence.

Additional examples of IGFALS mRNA sequences are readily available using publicly available databases, e.g., GenBank, UniProt, and OMIM.

“Insulin-like growth factor 1” or “IGF-1”, also known as MGF, encodes a protein similar to insulin in function and structure and is a member of a family of proteins involved in mediating growth and development. The encoded protein is processed from a precursor, bound by a specific receptor, and secreted. Defects in this gene are a cause of insulin-like growth factor I deficiency. Alternative splicing results in multiple transcript variants encoding different isoforms that may undergo similar processing to generate mature protein. Further information on IGF-1 is provided, for example, in the NCBI Gene database at www.ncbi.nlm.nih.gov/gene/3479 (which is incorporated herein by reference as of the date of filing this application).

As used herein, “insulin-like growth factor 1” is used interchangeably with the term “IGF-1” (and optionally any of the other recognized names listed above) refers to the naturally occurring gene that encodes an insulin-like growth factor 1 protein. The amino acid and complete coding sequences of the reference sequence of the human IGF-1 gene, transcript variant 1, mRNA, may be found in, for example, GenBank Accession No. GI: 930588898 (RefSeq Accession No. NM_001111283.2; SEQ ID NO: 11; SEQ ID NO: 12); human IGF-1 gene, transcript variant 4, mRNA, may be found at GenBank Accession No. GI: 930616505 (RefSeq Accession No. NM_000618.4; SEQ ID NO: 13 and SEQ ID NO:14); and human IGF-1, transcript variant 2, mRNA, may be found at GenBank Accession No. GI: 163659900 (RefSeq Accession No. NM_001111284.1; SEQ ID NO: 15 and 16. Mammalian orthologs of the human IGF-1 gene may be found in, for example, GI: 930155588 (RefSeq Accession No. NM 010512.5, mouseIGF-1; SEQ ID NO:17 and SEQ ID NO:18); GI: 126722710 (RefSeq Accession No. NM 001082478.1, rat; SEQ ID NO:19 and SEQ ID NO:20); GenBank Accession Nos. GI: 544472486 (RefSeq Accession No. XM_005572040.1, cynomolgus monkey; SEQ ID NO:21 and SEQ ID NO:22). Multiple sequence variants for each of the species are known.

A number of naturally occurring SNPs are known and can be found, for example, in the SNP database at the NCBI at www.ncbi.nlm.nih.gov/SNP/snp_ref.cgi?locusld=3479 (which is incorporated herein by reference as of the date of filing this application) which lists SNPs in human IGF-1. In preferred embodiments, such naturally occurring variants are included within the scope of the IGF-1 gene sequence.

Additional examples of IGF-1 mRNA sequences are readily available using publicly available databases, e.g., GenBank, UniProt, and OMIM.

As used herein, “target sequence” refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of an IGFALS gene or an IGF-1 gene, including mRNA that is a product of RNA processing of a primary transcription product. The target portion of the sequence will be at least long enough to serve as a substrate for iRNA-directed cleavage at or near that portion of the nucleotide sequence of an mRNA molecule formed during the transcription of an IGFALS gene or an IGF-1 gene gene. In one embodiment, the target sequence is within the protein coding region of IGFALS or IGF-1.

The target sequence may be from about 9-36 nucleotides in length, e.g., about 15-30 nucleotides in length. For example, the target sequence can be from about 15-30 nucleotides, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length. In some embodiments, the target sequence is about 19 to about 30 nucleotides in length. In other embodiments, the target sequence is about 19 to about 25 nucleotides in length. In still other embodiments, the target sequence is about 19 to about 23 nucleotides in length. In some embodiments, the target sequence is about 21 to about 23 nucleotides in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the invention.

As used herein, the term “strand comprising a sequence” refers to an oligonucleotide comprising a chain of nucleotides that is described by the sequence referred to using the standard nucleotide nomenclature.

“G,” “C,” “A,” “T,” and “U” each generally stand for a nucleotide that contains guanine, cytosine, adenine, thymidine, and uracil as a base, respectively. However, it will be understood that the term “ribonucleotide” or “nucleotide” can also refer to a modified nucleotide, as further detailed below, or a surrogate replacement moiety (see, e.g., Table 2). The skilled person is well aware that guanine, cytosine, adenine, and uracil can be replaced by other moieties without substantially altering the base pairing properties of an oligonucleotide comprising a nucleotide bearing such replacement moiety. For example, without limitation, a nucleotide comprising inosine as its base can base pair with nucleotides containing adenine, cytosine, or uracil. Hence, nucleotides containing uracil, guanine, or adenine can be replaced in the nucleotide sequences of dsRNA featured in the invention by a nucleotide containing, for example, inosine. In another example, adenine and cytosine anywhere in the oligonucleotide can be replaced with guanine and uracil, respectively to form G-U Wobble base pairing with the target mRNA. Sequences containing such replacement moieties are suitable for the compositions and methods featured in the invention.

The terms “iRNA,” “RNAi agent,” and “iRNA agent,” “RNA interference agent” as used interchangeably herein, refer to an agent that contains RNA as that term is defined herein, and which mediates the targeted cleavage of an RNA transcript via an RNA-induced silencing complex (RISC) pathway. iRNA directs the sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). The iRNA modulates, e.g., inhibits, the expression of the target gene, e.g., an IGFALS gene or an IGF-1 gene, in a cell, e.g., a cell within a subject, such as a mammalian subject.

In one embodiment, an RNAi agent of the invention includes a single stranded RNAi that interacts with a target RNA sequence, e.g., an IGFALS or IGF-1 target mRNA sequence, to direct the cleavage of the target RNA. Without wishing to be bound by theory it is believed that long double stranded RNA introduced into cells is broken down into double-stranded short interfering RNAs (siRNAs) comprising a sense strand and an antisense strand by a Type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). Dicer, a ribonuclease-III-like enzyme, processes these dsRNA into 19-23 base pair short interfering RNAs with characteristic two base 3′ overhangs (Bernstein, et al., (2001) Nature 409:363). These siRNAs are then incorporated into an RNA-induced silencing complex (RISC) where one or more helicases unwind the siRNA duplex, enabling the complementary antisense strand to guide target recognition (Nykanen, et al., (2001) Cell 107:309). Upon binding to the appropriate target mRNA, one or more endonucleases within the RISC cleave the target to induce silencing (Elbashir, et al., (2001) Genes Dev. 15:188). Thus, in one aspect the invention relates to a single stranded RNA (ssRNA) (the antisense strand of an siRNA duplex) generated within a cell and which promotes the formation of a RISC complex to effect silencing of the target gene, i.e., an IGFALS or IGF-1 gene. Accordingly, the term “siRNA” is also used herein to refer to an RNAi as described above.

In another embodiment, the RNAi agent may be a single-stranded RNA that is introduced into a cell or organism to inhibit a target mRNA. Single-stranded RNAi agents bind to the RISC endonuclease, Argonaute 2, which then cleaves the target mRNA. The single-stranded siRNAs are generally 15-30 nucleotides and are chemically modified. The design and testing of single-stranded RNAs are described in U.S. Pat. No. 8,101,348 and in Lima et al., (2012) Cell 150:883-894, the entire contents of each of which are hereby incorporated herein by reference. Any of the antisense nucleotide sequences described herein may be used as a single-stranded siRNA as described herein or as chemically modified by the methods described in Lima et al., (2012) Cell 150:883-894.

In certain embodiments, an “iRNA” for use in the compositions, uses, and methods of the invention is a double stranded RNA and is referred to herein as a “double stranded RNAi agent,” “double stranded RNA (dsRNA) molecule,” “dsRNA agent,” or “dsRNA”. The term “dsRNA”, refers to a complex of ribonucleic acid molecules, having a duplex structure comprising two anti-parallel and substantially complementary nucleic acid strands, referred to as having “sense” and “antisense” orientations with respect to a target RNA, i.e., an IGFALS gene or an IGF-1 gene. In some embodiments of the invention, a double stranded RNA (dsRNA) triggers the degradation of a target RNA, e.g., an mRNA, through a post-transcriptional gene-silencing mechanism referred to herein as RNA interference or RNAi.

In general, the majority of nucleotides of each strand of a dsRNA molecule are ribonucleotides, but as described in detail herein, each or both strands can also include one or more non-ribonucleotides, e.g., a deoxyribonucleotide or a modified nucleotide. In addition, as used in this specification, an “iRNA” may include ribonucleotides with chemical modifications; an iRNA may include substantial modifications at multiple nucleotides.

As used herein, the term “modified nucleotide” refers to a nucleotide having, independently, a modified sugar moiety, a modified internucleotide linkage, or modified nucleobase, or any combination thereof. Thus, the term modified nucleotide encompasses substitutions, additions or removal of, e.g., a functional group or atom, to internucleoside linkages, sugar moieties, or nucleobases. The modifications suitable for use in the agents of the invention include all types of modifications disclosed herein or known in the art. Any such modifications, as used in a siRNA type molecule, are encompassed by “iRNA” or “RNAi agent” for the purposes of this specification and claims.

The duplex region may be of any length that permits specific degradation of a desired target RNA through a RISC pathway, and may range from about 9 to 36 base pairs in length, e.g., about 15-30 base pairs in length, for example, about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 base pairs in length, such as about 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24,20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the invention.

The two strands forming the duplex structure may be different portions of one larger RNA molecule, or they may be separate RNA molecules. Where the two strands are part of one larger molecule, and therefore are connected by an uninterrupted chain of nucleotides between the 3′-end of one strand and the 5′-end of the respective other strand forming the duplex structure, the connecting RNA chain is referred to as a “hairpin loop.” A hairpin loop can comprise at least one unpaired nucleotide. In some embodiments, the hairpin loop can comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 23 or more unpaired nucleotides. In some embodiments, the hairpin loop can be 10 or fewer nucleotides. In some embodiments, the hairpin loop can be 8 or fewer unpaired nucleotides. In some embodiments, the hairpin loop can be 4-10 unpaired nucleotides. In some embodiments, the hairpin loop can be 4-8 nucleotides.

Where the two substantially complementary strands of a dsRNA are comprised by separate RNA molecules, those molecules need not, but can be covalently connected. Where the two strands are connected covalently by means other than an uninterrupted chain of nucleotides between the 3′-end of one strand and the 5′-end of the respective other strand forming the duplex structure, the connecting structure is referred to as a “linker.” The RNA strands may have the same or a different number of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of the dsRNA minus any overhangs that are present in the duplex. In addition to the duplex structure, an RNAi may comprise one or more nucleotide overhangs. In one embodiment of the RNAi agent, at least one strand comprises a 3′ overhang of at least 1 nucleotide. In another embodiment, at least one strand comprises a 3′ overhang of at least 2 nucleotides, e.g., 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In other embodiments, at least one strand of the RNAi agent comprises a 5′ overhang of at least 1 nucleotide. In certain embodiments, at least one strand comprises a 5′ overhang of at least 2 nucleotides, e.g., 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In still other embodiments, both the 3′ and the 5′ end of one strand of the RNAi agent comprise an overhang of at least 1 nucleotide.

In certain embodiments, an iRNA agent of the invention is a dsRNA, each strand of which comprises 19-23 nucleotides, that interacts with a target RNA sequence, e.g., an IGFALS gene or an IGF-1 gene. Without wishing to be bound by theory, long double stranded RNA introduced into cells is broken down into siRNA by a Type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). Dicer, a ribonuclease-III-like enzyme, processes the dsRNA into 19-23 base pair short interfering RNAs with characteristic two base 3′ overhangs (Bernstein, et al., (2001) Nature 409:363). The siRNAs are then incorporated into an RNA-induced silencing complex (RISC) where one or more helicases unwind the siRNA duplex, enabling the complementary antisense strand to guide target recognition (Nykanen, et al., (2001) Cell 107:309). Upon binding to the appropriate target mRNA, one or more endonucleases within the RISC cleave the target to induce silencing (Elbashir, et al., (2001) Genes Dev. 15:188).

In some embodiments, an iRNA of the invention is a dsRNA of 24-30 nucleotides, or possibly even longer, e.g., 25-35, 27-53, or 27-49 nucleotides, that interacts with a target RNA sequence, e.g., an IGFALS target mRNA sequence or an IGF-1 target mRNA sequence, to direct the cleavage of the target RNA. Without wishing to be bound by theory, long double stranded RNA introduced into cells is broken down into siRNA by a Type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). Dicer, a ribonuclease-III-like enzyme, processes the dsRNA into 19-23 base pair short interfering RNAs with characteristic two base 3′ overhangs (Bernstein, et al., (2001) Nature 409:363). The siRNAs are then incorporated into an RNA-induced silencing complex (RISC) where one or more helicases unwind the siRNA duplex, enabling the complementary antisense strand to guide target recognition (Nykanen, et al., (2001) Cell 107:309). Upon binding to the appropriate target mRNA, one or more endonucleases within the RISC cleave the target to induce silencing (Elbashir, et al., (2001) Genes Dev. 15:188).

As used herein, the term “nucleotide overhang” refers to at least one unpaired nucleotide that protrudes from the duplex structure of a double stranded iRNA. For example, when a 3′-end of one strand of a dsRNA extends beyond the 5′-end of the other strand, or vice versa, there is a nucleotide overhang. A dsRNA can comprise an overhang of at least one nucleotide; alternatively the overhang can comprise at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides or more. A nucleotide overhang can comprise or consist of a nucleotide/nucleoside analog, including a deoxynucleotide/nucleoside. The overhang(s) can be on the sense strand, the antisense strand, or any combination thereof. Furthermore, the nucleotide(s) of an overhang can be present on the 5′-end, 3′-end, or both ends of either an antisense or sense strand of a dsRNA. In one embodiment of the dsRNA, at least one strand comprises a 3′ overhang of at least 1 nucleotide. In another embodiment, at least one strand comprises a 3′ overhang of at least 2 nucleotides, e.g., 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In other embodiments, at least one strand of the RNAi agent comprises a 5′ overhang of at least 1 nucleotide. In certain embodiments, at least one strand comprises a 5′ overhang of at least 2 nucleotides, e.g., 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In still other embodiments, both the 3′ and the 5′ end of one strand of the RNAi agent comprise an overhang of at least 1 nucleotide.

In certain embodiments, the antisense strand of a dsRNA has a 1-10 nucleotide, e.g., 0-3, 1-3, 2-4, 2-5, 4-10, 5-10, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide, overhang at the 3′-end or the 5′-end. In certain embodiments, the overhang on the sense strand or the antisense strand, or both, can include extended lengths longer than 10 nucleotides, e.g., 1-30 nucleotides, 2-30 nucleotides, 10-30 nucleotides, or 10-15 nucleotides in length. In certain embodiments, an extended overhang is on the sense strand of the duplex. In certain embodiments, an extended overhang is present on the 3′end of the sense strand of the duplex. In certain embodiments, an extended overhang is present on the 5′end of the sense strand of the duplex. In certain embodiments, an extended overhang is on the antisense strand of the duplex. In certain embodiments, an extended overhang is present on the 3′end of the antisense strand of the duplex. In certain embodiments, an extended overhang is present on the 5′end of the antisense strand of the duplex. In certain embodiments, one or more of the nucleotides in the overhang is replaced with a nucleoside thiophosphate.

“Blunt” or “blunt end” means that there are no unpaired nucleotides at that end of the double stranded RNAi agent, i.e., no nucleotide overhang. A “blunt ended” double stranded RNAi agent is double stranded over its entire length, i.e., no nucleotide overhang at either end of the molecule. The RNAi agents of the invention include RNAi agents with no nucleotide overhang at one end (i.e., agents with one overhang and one blunt end) or with no nucleotide overhangs at either end.

The term “antisense strand” or “guide strand” refers to the strand of an iRNA, e.g., a dsRNA, which includes a region that is substantially complementary to a target sequence, e.g., an IGFALS or IGF-1 mRNA. As used herein, the term “region of complementarity” refers to the region on the antisense strand that is substantially complementary to a sequence, for example a target sequence, e.g., an IGFALS or IGF-1 nucleotide sequence, as defined herein. Where the region of complementarity is not fully complementary to the target sequence, the mismatches can be in the internal or terminal regions of the molecule. Generally, the most tolerated mismatches are in the terminal regions, e.g., within 5, 4, 3, 2, or 1 nucleotides of the 5′- or 3′-end of the iRNA. In some embodiments, a double stranded RNAi agent of the invention includes a nucleotide mismatch in the antisense strand. In some embodiments, a double stranded RNAi agent of the invention includes a nucleotide mismatch in the sense strand. In some embodiments, the nucleotide mismatch is, for example, within 5, 4, 3, 2, or 1 nucleotides from the 3′-end of the iRNA. In another embodiment, the nucleotide mismatch is, for example, in the 3′-terminal nucleotide of the iRNA.

The term “sense strand” or “passenger strand” as used herein, refers to the strand of an iRNA that includes a region that is substantially complementary to a region of the antisense strand as that term is defined herein.

As used herein, “substantially all of the nucleotides are modified” are largely but not wholly modified and can include not more than 5, 4, 3, 2, or 1 unmodified nucleotides.

As used herein, the term “cleavage region” refers to a region that is located immediately adjacent to the cleavage site. The cleavage site is the site on the target at which cleavage occurs. In some embodiments, the cleavage region comprises three bases on either end of, and immediately adjacent to, the cleavage site. In some embodiments, the cleavage region comprises two bases on either end of, and immediately adjacent to, the cleavage site. In some embodiments, the cleavage site specifically occurs at the site bound by nucleotides 10 and 11 of the antisense strand, and the cleavage region comprises nucleotides 11, 12 and 13.

As used herein, and unless otherwise indicated, the term “complementary,” when used to describe a first nucleotide sequence in relation to a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize and form a duplex structure under certain conditions with an oligonucleotide or polynucleotide comprising the second nucleotide sequence, as will be understood by the skilled person. Such conditions can, for example, be stringent conditions, where stringent conditions can include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50° C. or 70° C. for 12-16 hours followed by washing (see, e.g., “Molecular Cloning: A Laboratory Manual, Sambrook, et al. (1989) Cold Spring Harbor Laboratory Press). Other conditions, such as physiologically relevant conditions as can be encountered inside an organism, can apply. The skilled person will be able to determine the set of conditions most appropriate for a test of complementarity of two sequences in accordance with the ultimate application of the hybridized nucleotides.

Complementary sequences within an iRNA, e.g., within a dsRNA as described herein, include base-pairing of the oligonucleotide or polynucleotide comprising a first nucleotide sequence to an oligonucleotide or polynucleotide comprising a second nucleotide sequence over the entire length of one or both nucleotide sequences. Such sequences can be referred to as “fully complementary” with respect to each other herein. However, where a first sequence is referred to as “substantially complementary” with respect to a second sequence herein, the two sequences can be fully complementary, or they can form one or more, but generally not more than 5, 4, 3, or 2 mismatched base pairs upon hybridization for a duplex up to 30 base pairs, while retaining the ability to hybridize under the conditions most relevant to their ultimate application, e.g., inhibition of gene expression via a RISC pathway. However, where two oligonucleotides are designed to form, upon hybridization, one or more single stranded overhangs, such overhangs shall not be regarded as mismatches with regard to the determination of complementarity. For example, a dsRNA comprising one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length, wherein the longer oligonucleotide comprises a sequence of 21 nucleotides that is fully complementary to the shorter oligonucleotide, can yet be referred to as “fully complementary” for the purposes described herein.

“Complementary” sequences, as used herein, can also include, or be formed entirely from, non-Watson-Crick base pairs or base pairs formed from non-natural and modified nucleotides, in so far as the above requirements with respect to their ability to hybridize are fulfilled. Such non-Watson-Crick base pairs include, but are not limited to, G:U Wobble or Hoogstein base pairing.

The terms “complementary,” “fully complementary” and “substantially complementary” herein can be used with respect to the base matching between the sense strand and the antisense strand of a dsRNA, or between the antisense strand of a double stranded RNAi agent and a target sequence, as will be understood from the context of their use.

As used herein, a polynucleotide that is “substantially complementary to at least part of” a messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a contiguous portion of the mRNA of interest (e.g., an mRNA encoding an IGFALS gene or an IGF-1 gene). For example, a polynucleotide is complementary to at least a part of an IGFALS or IGF-1 mRNA if the sequence is substantially complementary to a non-interrupted portion of an mRNA encoding an IGFALS or IGF-1 gene.

Accordingly, in some embodiments, the sense strand polynucleotides and the antisense polynucleotides disclosed herein are fully complementary to the target IGFALS or IGF-1 sequence.

In one embodiment, the antisense polynucleotides disclosed herein are fully complementary to the target IGFALS sequence. In other embodiments, the antisense polynucleotides disclosed herein are substantially complementary to the target IGFALS sequence and comprise a contiguous nucleotide sequence which is at least about 80% complementary over its entire length to the equivalent region of the nucleotide sequence of any one of SEQ ID NOs:1, 3, 5, 7, or 9, or a fragment of any one of SEQ ID NOs:1, 3, 5, 7, or 9, such as about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about % 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary.

In other embodiments, the antisense polynucleotides disclosed herein are substantially complementary to the target IGFALS sequence and comprise a contiguous nucleotide sequence which is at least about 80% complementary over its entire length to any one of the sense strand nucleotide sequences in any one of Tables 3, 5, 6, 8, 12, or 14, or a fragment of any one of the sense strand nucleotide sequences in any one of Tables 3, 5, 6, 8, 12, or 14, such as about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about % 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary.

In one embodiment, an RNAi agent of the invention includes a sense strand that is substantially complementary to an antisense polynucleotide which, in turn, is complementary to a target IGFALS sequence and comprises a contiguous nucleotide sequence which is at least about 80% complementary over its entire length to any one of the sense strand nucleotide sequences in any one of Tables 3, 5, 6, 8, 12, or 14, or a fragment of any one of the sense strand nucleotide sequences in any one of Tables 3, 5, 6, 8, 12, or 14, such as about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about % 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary.

In one embodiment, the antisense polynucleotides disclosed herein are fully complementary to the target IGF-1 sequence. In other embodiments, the antisense polynucleotides disclosed herein are substantially complementary to the target IGF-1 sequence and comprise a contiguous nucleotide sequence which is at least about 80% complementary over its entire length to the equivalent region of the nucleotide sequence of any one of SEQ ID NOs:11, 13, 15, 17, 19, or 21, or a fragment of any one of SEQ ID NOs:11, 13, 15, 17, 19, or 21, such as about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about % 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary.

In other embodiments, the antisense polynucleotides disclosed herein are substantially complementary to the target IGF-1 sequence and comprise a contiguous nucleotide sequence which is at least about 80% complementary over its entire length to any one of the sense strand nucleotide sequences in any one of Tables 9, 11, 15, 17, 18, or 20, or a fragment of any one of the sense strand nucleotide sequences in any one of Tables 9, 11, 15, 17, 18, or 20, such as about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about % 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary.

In one embodiment, an RNAi agent of the invention includes a sense strand that is substantially complementary to an antisense polynucleotide which, in turn, is complementary to a target IGF-1 sequence and comprises a contiguous nucleotide sequence which is at least about 80% complementary over its entire length to any one of the sense strand nucleotide sequences in any one of Tables 9, 11, 15, 17, 18, or 20, or a fragment of any one of the sense strand nucleotide sequences in any one of Tables 9, 11, 15, 17, 18, or 20, such as about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about % 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary.

In an aspect of the invention, an agent for use in the methods and compositions of the invention is a single-stranded antisense oligonucleotide molecule that inhibits a target mRNA via an antisense inhibition mechanism. The single-stranded antisense oligonucleotide molecule is complementary to a sequence within the target mRNA. The single-stranded antisense oligonucleotides can inhibit translation in a stoichiometric manner by base pairing to the mRNA and physically obstructing the translation machinery, see Dias, N. et al., (2002) Mol Cancer Ther 1:347-355. The single-stranded antisense oligonucleotide molecule may be about 14 to about 30 nucleotides in length and have a sequence that is complementary to a target sequence. For example, the single-stranded antisense oligonucleotide molecule may comprise a sequence that is at least 14, 15, 16, 17, 18, 19, 20, or more contiguous nucleotides from any one of the antisense sequences described herein.

The phrase “contacting a cell with an iRNA,” such as a dsRNA, as used herein, includes contacting a cell by any possible means. Contacting a cell with an iRNA includes contacting a cell in vitro with the iRNA or contacting a cell in vivo with the iRNA. The contacting may be done directly or indirectly. Thus, for example, the iRNA may be put into physical contact with the cell by the individual performing the method, or alternatively, the iRNA may be put into a situation that will permit or cause it to subsequently come into contact with the cell.

Contacting a cell in vitro may be done, for example, by incubating the cell with the iRNA. Contacting a cell in vivo may be done, for example, by injecting the iRNA into or near the tissue where the cell is located, or by injecting the iRNA into another area, e.g., the bloodstream or the subcutaneous space, such that the agent will subsequently reach the tissue where the cell to be contacted is located. For example, the iRNA may contain or be coupled to a ligand, e.g., GalNAc3, that directs the iRNA to a site of interest, e.g., the liver. Combinations of in vitro and in vivo methods of contacting are also possible. For example, a cell may also be contacted in vitro with an iRNA and subsequently transplanted into a subject.

In certain embodiments, contacting a cell with an iRNA includes “introducing” or “delivering the iRNA into the cell” by facilitating or effecting uptake or absorption into the cell. Absorption or uptake of an iRNA can occur through unaided diffusion or active cellular processes, or by auxiliary agents or devices. Introducing an iRNA into a cell may be in vitro or in vivo. For example, for in vivo introduction, iRNA can be injected into a tissue site or administered systemically. In vivo delivery can also be done by a beta-glucan delivery system, such as those described in U.S. Pat. Nos. 5,032,401 and 5,607,677, and US Publication No. 2005/0281781, the entire contents of which are hereby incorporated herein by reference. In vitro introduction into a cell includes methods known in the art such as electroporation and lipofection. Further approaches are described herein below or are known in the art.

The term “lipid nanoparticle” or “LNP” is a vesicle comprising a lipid layer encapsulating a pharmaceutically active molecule, such as a nucleic acid molecule, e.g., an iRNA or a plasmid from which an iRNA is transcribed. LNPs are described in, for example, U.S. Pat. Nos. 6,858,225, 6,815,432, 8,158,601, and 8,058,069, the entire contents of which are hereby incorporated herein by reference.

As used herein, a “subject” is an animal, such as a mammal, including a primate (such as a human, a non-human primate, e.g., a monkey, and a chimpanzee), a non-primate (such as a cow, a pig, a camel, a llama, a horse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a dog, a rat, a mouse, a horse, and a whale), or a bird (e.g., a duck or a goose) that expresses the target gene, either endogenously or heterologously. It is understood that the sequence of the PHD gene must be sufficiently complementary to the antisense strand of the iRNA agent for the agent to be used in the indicated species. In certain embodiments, the subject is a human, such as a human being treated or assessed for a disease, disorder or condition that would benefit from reduction in an IGFALS gene or an IGF-1 gene expression or replication; a human at risk for a disease, disorder or condition that would benefit from reduction in IGFALS or IGF-1 gene expression; a human having a disease, disorder or condition that would benefit from reduction in IGFALS or IGF-1 gene expression; or human being treated for a disease, disorder or condition that would benefit from reduction in IGFALS or IGF-1 gene expression, as described herein. In some embodiments, the subject is a female human. In other embodiments, the subject is a male human.

As used herein, the terms “treating” or “treatment” refer to a beneficial or desired result including, but not limited to, alleviation or amelioration of one or more symptoms associated with IGFALS or IGF-1 gene expression or IGFALS or IGF-1 protein production, e.g., acromegaly, cancer. “Treatment” can also mean prolonging survival as compared to expected survival in the absence of treatment.

The term “lower” or “reduce” in the context of the level of IGFALS or IGF-1 gene expression or IGFALS or IGF-1 protein production in a subject, or a disease marker or symptom refers to a statistically significant decrease in such level. The decrease can be, for example, at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or below the level of detection for the detection method. In certain embodiments, the decrease is down to a level accepted as within the range of normal for an individual without such disorder which can also be referred to as a normalization of a level. For example, lowering cholesterol to 180 mg/dl or lower would be considered to be within the range of normal for a subject. A subject having a cholesterol level of 230 mg/dl with a cholesterol level decreased to 210 mg/dl would have a cholesterol level that was decreased by 40% (230−210/230−180=20/50=40% reduction). In certain embodiments, the reduction is the normalization of the level of a sign or symptom of a disease, a reduction in the difference between the subject level of a sign of the disease and the normal level of the sign for the disease (e.g., the upper level of normal when the level must be reduced to reach a normal level, and the lower level of normal when the level must be increased to reach a normal level). In certain embodiments, the methods include a clinically relevant inhibition of expression of IGFALS or IGF-1, e.g. as demonstrated by a clinically relevant outcome after treatment of a subject with an agent to reduce the expression of IGFALS or IGF-1.

As used herein, “prevention” or “preventing,” when used in reference to a disease, disorder or condition thereof, that would benefit from a reduction in expression of an IGFALS gene or an IGF-1 gene or production of an IGFALS or an IGF-1 protein, refers to a reduction in the likelihood that a subject will develop a symptom associated with such a disease, disorder, or condition, e.g., a symptom of IGFALS or IGF-1 gene expression, such as the presence of elevated levels of proteins in the IGF signaling pathway, e.g., acromegaly or cancer. The failure to develop a disease, disorder or condition, or the reduction in the development of a symptom or comorbidity associated with such a disease, disorder or condition (e.g., by at least about 10% on a clinically accepted scale for that disease or disorder), or the exhibition of delayed symptoms or disease progression (e.g., delayed cancer progression as determined using RECIST criteria) by days, weeks, months or years is considered effective prevention. Prevention may require the administration of more than one dose.

As used herein, the term “IGF system-associated disease,” used interchangeable with the terms “insulin-like growth factor binding protein, acid labile subunit-associated disease,” “IGFALS-associated disease,” “IGF-associated disease,” or “IGF-1-associated disease” is a disease or disorder that is caused by, or associated with IGFALS or IGF gene expression or IGFALS or IGF protein production. The term “IGF system-associated disease” includes a disease, disorder or condition that would benefit from a decrease in IGFALS or IGF-1 gene expression, replication, or protein activity. Non-limiting examples of IGF system-associated diseases include, for example, acromegaly, gigantism, and cancer, especially metastatic cancer.

In certain embodiments, an IGF system-associated disease-associated disease is acromegaly.

“Therapeutically effective amount,” as used herein, is intended to include the amount of an iRNA that, when administered to a patient for treating a subject having acromealgy, cancer, or IGF system-associated disease, is sufficient to effect treatment of the disease (e.g., by diminishing, ameliorating or maintaining the existing disease or one or more symptoms of disease or its related comorbidities). The “therapeutically effective amount” may vary depending on the iRNA, how it is administered, the disease and its severity and the history, age, weight, family history, genetic makeup, stage of pathological processes mediated by IGFALS or IGF-1 gene expression, the types of preceding or concomitant treatments, if any, and other individual characteristics of the patient to be treated. Treatment may require the administration of more than one dose.

“Prophylactically effective amount,” as used herein, is intended to include the amount of an iRNA that, when administered to a subject who does not yet experience or display symptoms of acromealgy, cancer, or other IGF system-associated disease-associated diseases, but who may be predisposed to an IGF system-associated disease-associated disease, is sufficient to prevent or delay the development or progression of the disease or one or more symptoms of the disease for a clinically significant period of time. The “prophylactically effective amount” may vary depending on the iRNA, how it is administered, the degree of risk of disease, and the history, age, weight, family history, genetic makeup, the types of preceding or concomitant treatments, if any, and other individual characteristics of the patient to be treated.

A “therapeutically-effective amount” or “prophylacticaly effective amount” also includes an amount of an iRNA that produces some desired local or systemic effect at a reasonable benefit/risk ratio applicable to any treatment. iRNAs employed in the methods of the present invention may be administered in a sufficient amount to produce a reasonable benefit/risk ratio applicable to such treatment.

The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human subjects and animal subjects without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically-acceptable carrier” as used herein means a pharmaceutically-acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject being treated. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium state, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids (23) serum component, such as serum albumin, HDL and LDL; and (22) other non-toxic compatible substances employed in pharmaceutical formulations.

The term “sample,” as used herein, includes a collection of similar fluids, cells, or tissues isolated from a subject, as well as fluids, cells, or tissues present within a subject. Examples of biological fluids include blood, serum and serosal fluids, plasma, cerebrospinal fluid, ocular fluids, lymph, urine, saliva, and the like. Tissue samples may include samples from tissues, organs, or localized regions. For example, samples may be derived from particular organs, parts of organs, or fluids or cells within those organs. In certain embodiments, samples may be derived from the liver (e.g., whole liver or certain segments of liver or certain types of cells in the liver, such as, e.g., hepatocytes). A “sample derived from a subject” can refer to blood drawn from the subject, or plasma derived therefrom. In certain embodiments when detecting a level of IGF-1, a “sample” preferably refers to a tissue or body fluid from a subject in which IGF-1 is detectable prior to administration of an agent of the invention, e.g., a liver biopsy from a subject with a acromegaly, a tumor. In certain subjects, e.g., healthy subjects, the level of IGF-1 may not be detectable in a number of body fluids, cell types, and tissues.

I. iRNAs of the Invention

The present invention provides iRNAs which inhibit the expression of an IGFALS gene or an IGF-1 gene. In preferred embodiments, the iRNA includes double stranded ribonucleic acid (dsRNA) molecules for inhibiting the expression of an IGFALS gene or an IGF-1 gene in a cell, such as a cell within a subject, e.g., a mammal, such as a human having an IGF system-associated disease-associated disease, e.g., acromeagly. The dsRNAi agent includes an antisense strand having a region of complementarity which is complementary to at least a part of an mRNA formed in the expression of an IGFALS gene or an IGF-1 gene. The region of complementarity is about 30 nucleotides or less in length (e.g., about 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, or 18 nucleotides or less in length). Upon contact with a cell expressing the IGFALS gene or the IGF-1 gene, the iRNA inhibits the expression of the IGFALS gene or the IGF-1 gene (e.g., a human, a primate, a non-primate, or a bird IGFALS gene or IGF-1 gene) by at least 20%, preferably at least 30%, as assayed by, for example, a PCR or branched DNA (bDNA)-based method, or by a protein-based method, such as by immunofluorescence analysis, using, for example, western blotting or flowcytometric techniques. In preferred embodiments, inhibiton of expression is determined by the qPCR method provided in the examples. For in vitro assessment of activity, percent inhibition is determined using the methods provided in Example 2 at a single dose at a 10 nM duplex final concentration. For in vivo studies, the level after treatment can be compared to, for example, an appropriate historical control or a pooled population sample control to determine the level of reduction, e.g., when a baseline value is no available for the subject.

A dsRNA includes two RNA strands that are complementary and hybridize to form a duplex structure under conditions in which the dsRNA will be used. One strand of a dsRNA (the antisense strand) includes a region of complementarity that is substantially complementary, and generally fully complementary, to a target sequence. The target sequence can be derived from the sequence of an mRNA formed during the expression of an IGFALS gene or IGF-1 gene. The other strand (the sense strand) includes a region that is complementary to the antisense strand, such that the two strands hybridize and form a duplex structure when combined under suitable conditions. As described elsewhere herein and as known in the art, the complementary sequences of a dsRNA can also be contained as self-complementary regions of a single nucleic acid molecule, as opposed to being on separate oligonucleotides.

Generally, the duplex structure is about 15 to 30 base pairs in length, e.g., 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the invention.

Similarly, the region of complementarity to the target sequence is about 15 to 30 nucleotides in length, e.g., 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the invention.

In some embodiments, the dsRNA is about 15 to 23 nucleotides in length, or about 25 to 30 nucleotides in length. In general, the dsRNA is long enough to serve as a substrate for the Dicer enzyme. For example, it is well-known in the art that dsRNAs longer than about 21-23 nucleotides in length may serve as substrates for Dicer. As the ordinarily skilled person will also recognize, the region of an RNA targeted for cleavage will most often be part of a larger RNA molecule, often an mRNA molecule. Where relevant, a “part” of an mRNA target is a contiguous sequence of an mRNA target of sufficient length to allow it to be a substrate for RNAi-directed cleavage (i.e., cleavage through a RISC pathway).

One of skill in the art will also recognize that the duplex region is a primary functional portion of a dsRNA, e.g., a duplex region of about 9 to about 36 base pairs, e.g., 10-36, 11-36, 12-36, 13-36, 14-36, 15-36, 9-35, 10-35, 11-35, 12-35, 13-35, 14-35, 15-35, 9-34, 10-34, 11-34, 12-34, 13-34, 14-34, 15-34, 9-33, 10-33, 11-33, 12-33, 13-33, 14-33, 15-33, 9-32, 10-32, 11-32, 12-32, 13-32, 14-32, 15-32, 9-31, 10-31, 11-31, 12-31, 13-32, 14-31, 15-31, 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs. Thus, in one embodiment, to the extent that it becomes processed to a functional duplex, of e.g., 15-30 base pairs, that targets a desired RNA for cleavage, an RNA molecule or complex of RNA molecules having a duplex region greater than 30 base pairs is a dsRNA. Thus, an ordinarily skilled artisan will recognize that in one embodiment, a miRNA is a dsRNA. In another embodiment, a dsRNA is not a naturally occurring miRNA. In another embodiment, an iRNA agent useful to target IGFALS or IGF-1 gene expression is not generated in the target cell by cleavage of a larger dsRNA.

A dsRNA as described herein can further include one or more single-stranded nucleotide overhangs e.g., 1-4, 2-4, 1-3, 2-3, 1, 2, 3, or 4 nucleotides. dsRNAs having at least one nucleotide overhang can have superior inhibitory properties relative to their blunt-ended counterparts. A nucleotide overhang can comprise or consist of a nucleotide/nucleoside analog, including a deoxynucleotide/nucleoside. The overhang(s) can be on the sense strand, the antisense strand, or any combination thereof. Furthermore, the nucleotide(s) of an overhang can be present on the 5′-end, 3′-end, or both ends of an antisense or sense strand of a dsRNA.

A dsRNA can be synthesized by standard methods known in the art as further discussed below, e.g., by use of an automated DNA synthesizer, such as are commercially available from, for example, Biosearch, Applied Biosystems, Inc.

Double stranded RNAi compounds of the invention may be prepared using a two-step procedure. First, the individual strands of the double stranded RNA molecule are prepared separately. Then, the component strands are annealed. The individual strands of the siRNA compound can be prepared using solution-phase or solid-phase organic synthesis or both. Organic synthesis offers the advantage that the oligonucleotide strands comprising unnatural or modified nucleotides can be easily prepared. Similarly, single-stranded oligonucleotides of the invention can be prepared using solution-phase or solid-phase organic synthesis or both.

In an aspect, a dsRNA of the invention for inhibing the expression of an IGFALS gene includes at least two nucleotide sequences, a sense sequence and an anti-sense sequence. The sense strand is selected from the group of sequences provided in any one of Tables 3, 5, 6, 8, 12, and 14, and the corresponding antisense strand of the sense strand is selected from the group of sequences in any one of Tables 3, 5, 6, 8, 12, and 14. In this aspect, one of the two sequences is complementary to the other of the two sequences, with one of the sequences being substantially complementary to a sequence of an mRNA generated in the expression of an IGFALS gene. As such, in this aspect, a dsRNA will include two oligonucleotides, where one oligonucleotide is described as the sense strand in any one of Table 3, 5, 6, 8, 12, and 14, and the second oligonucleotide is described as the corresponding antisense strand of the sense strand in any one of Table 3, 5, 6, 8, 12, and 14. In certain embodiments, the substantially complementary sequences of the dsRNA are contained on separate oligonucleotides. In other embodiments, the substantially complementary sequences of the dsRNA are contained on a single oligonucleotide.

In an aspect, a dsRNA of the invention for inhibing the expression of an IGF-1 gene includes at least two nucleotide sequences, a sense sequence and an anti-sense sequence. The sense strand is selected from the group of sequences provided in any one of Tables 9, 11, 15, 17, 18, and 20, and the corresponding antisense strand of the sense strand is selected from the group of sequences in any one of Tables 9, 11, 15, 17, 18, and 20. In this aspect, one of the two sequences is complementary to the other of the two sequences, with one of the sequences being substantially complementary to a sequence of an mRNA generated in the expression of an IGF-1 gene. As such, in this aspect, a dsRNA will include two oligonucleotides, where one oligonucleotide is described as the sense strand in any one of Table 9, 11, 15, 17, 18, and 20, and the second oligonucleotide is described as the corresponding antisense strand of the sense strand in any one of Table 9, 11, 15, 17, 18, and 20. In certain embodiments, the substantially complementary sequences of the dsRNA are contained on separate oligonucleotides. In other embodiments, the substantially complementary sequences of the dsRNA are contained on a single oligonucleotide.

It will be understood that, although the sequences in Tables 3, 6, 9, 12, 15, and 18 are not described as modified or conjugated sequences, the RNA of the iRNA of the invention e.g., a dsRNA of the invention, may comprise any one of the sequences set forth in any one of Tables 3, 6, 9, 12, 15, and 18, or the sequences of any one of Tables 5, 8, 11, 14, 17, and 20 that are modified, or the sequences of any one of Tables 5, 8, 11, 14, 17, and 20 that are conjugated to a ligand. In other words, the invention encompasses dsRNAs of any one of Tables 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, and 20 which are un-modified, un-conjugated, modified, or conjugated, as described herein.

The skilled person is well aware that dsRNAs having a duplex structure of between about 20 and 23 base pairs, e.g., 21, base pairs have been hailed as particularly effective in inducing RNA interference (Elbashir et al., EMBO 2001, 20:6877-6888). However, others have found that shorter or longer RNA duplex structures can also be effective (Chu and Rana (2007) RNA 14:1714-1719; Kim et al. (2005) Nat Biotech 23:222-226). In the embodiments described above, by virtue of the nature of the oligonucleotide sequences provided in any one of Tables 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, and 20, dsRNAs described herein can include at least one strand of a length of minimally 21 nucleotides. It can be reasonably expected that shorter duplexes having one of the sequences of Tables 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, and 20 minus only a few nucleotides on one or both ends can be similarly effective as compared to the dsRNAs described above. Hence, dsRNAs having a sequence of at least 15, 16, 17, 18, 19, 20, or more contiguous nucleotides derived from one of the sequences of Tables 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, and 20, and differing in their ability to inhibit the expression of an IGFALS gene or an IGF-1 gene by not more than about 5, 10, 15, 20, 25, or 30% inhibition from a dsRNA comprising the full sequence, are contemplated to be within the scope of the present invention.

In addition, the RNAs provided in Tables 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, and 20 identify a site(s) in an IGFALS transcript or IGF-1 transcript that is susceptible to RISC-mediated cleavage. As such, the present invention further features iRNAs that target within one of these sites. As used herein, an iRNA is said to target within a particular site of an RNA transcript if the iRNA promotes cleavage of the transcript anywhere within that particular site. Such an iRNA will generally include at least about 15 contiguous nucleotides from one of the sequences provided in Tables 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, and 20 coupled to additional nucleotide sequences taken from the region contiguous to the selected sequence in an IGFALS gene or an IGF-1 gene.

While a target sequence is generally about 15-30 nucleotides in length, there is wide variation in the suitability of particular sequences in this range for directing cleavage of any given target RNA. Various software packages and the guidelines set out herein provide guidance for the identification of optimal target sequences for any given gene target, but an empirical approach can also be taken in which a “window” or “mask” of a given size (as a non-limiting example, 21 nucleotides) is literally or figuratively (including, e.g., in silico) placed on the target RNA sequence to identify sequences in the size range that can serve as target sequences. By moving the sequence “window” progressively one nucleotide upstream or downstream of an initial target sequence location, the next potential target sequence can be identified, until the complete set of possible sequences is identified for any given target size selected. This process, coupled with systematic synthesis and testing of the identified sequences (using assays as described herein or as known in the art or provided herein) to identify those sequences that perform optimally can identify those RNA sequences that, when targeted with an iRNA agent, mediate the best inhibition of target gene expression. Thus, while the sequences identified, for example, in Tables 3 and 5 represent effective target sequences, it is contemplated that further optimization of inhibition efficiency can be achieved by progressively “walking the window” one nucleotide upstream or downstream of the given sequences to identify sequences with equal or better inhibition characteristics.

Further, it is contemplated that for any sequence identified, e.g., in Tables 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, and 20, further optimization could be achieved by systematically either adding or removing nucleotides to generate longer or shorter sequences and testing those sequences generated by walking a window of the longer or shorter size up or down the target RNA from that point. Again, coupling this approach to generating new candidate targets with testing for effectiveness of iRNAs based on those target sequences in an inhibition assay as known in the art or as described herein can lead to further improvements in the efficiency of inhibition. Further still, such optimized sequences can be adjusted by, e.g., the introduction of modified nucleotides as described herein or as known in the art, addition or changes in overhang, or other modifications as known in the art or discussed herein to further optimize the molecule (e.g., increasing serum stability or circulating half-life, increasing thermal stability, enhancing transmembrane delivery, targeting to a particular location or cell type, increasing interaction with silencing pathway enzymes, increasing release from endosomes) as an expression inhibitor.

An iRNA as described herein can contain one or more mismatches to the target sequence. In one embodiment, an iRNA as described herein contains no more than 3 mismatches. If the antisense strand of the iRNA contains mismatches to a target sequence, it is preferable that the area of mismatch is not located in the center of the region of complementarity. If the antisense strand of the iRNA contains mismatches to the target sequence, it is preferable that the mismatch be restricted to be within the last 5 nucleotides from either the 5′- or 3′-end of the region of complementarity. For example, for a 23 nucleotide iRNA agent the strand which is complementary to a region of an IGFALS gene or an IGF-1 gene, generally does not contain any mismatch within the central 13 nucleotides. The methods described herein or methods known in the art can be used to determine whether an iRNA containing a mismatch to a target sequence is effective in inhibiting the expression of an IGFALS gene or IGF-1 gene. Consideration of the efficacy of iRNAs with mismatches in inhibiting expression of an IGFALS gene or an IGF-1 gene is important, especially if the particular region of complementarity in an IGFALS gene or an IGF-1 gene is known to have polymorphic sequence variation within the population.

II. Modified iRNAs of the Invention

In certain embodiments, the RNA of the iRNA of the invention e.g., a dsRNA, is unmodified, and does not comprise, e.g., chemical modifications or conjugations known in the art and described herein. In other embodiments, the RNA of an iRNA of the invention, e.g., a dsRNA, is chemically modified to enhance stability or other beneficial characteristics. In certain embodiments of the invention, substantially all of the nucleotides of an iRNA of the invention are modified. In other embodiments of the invention, all of the nucleotides of an iRNA or substantially all of the nucleotides of an iRNA are modified, i.e., not more than 5, 4, 3, 2, or 1 unmodified nucleotides are present in a strand of the iRNA.

The nucleic acids featured in the invention can be synthesized or modified by methods well established in the art, such as those described in “Current protocols in nucleic acid chemistry,” Beaucage, S. L. et al. (Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA, which is hereby incorporated herein by reference. Modifications include, for example, end modifications, e.g., 5′-end modifications (phosphorylation, conjugation, inverted linkages) or 3′-end modifications (conjugation, DNA nucleotides, inverted linkages, etc.); base modifications, e.g., replacement with stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, removal of bases (abasic nucleotides), or conjugated bases; sugar modifications (e.g., at the 2′-position or 4′-position) or replacement of the sugar; or backbone modifications, including modification or replacement of the phosphodiester linkages. Specific examples of iRNA compounds useful in the embodiments described herein include, but are not limited to RNAs containing modified backbones or no natural internucleoside linkages. RNAs having modified backbones include, among others, those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified RNAs that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides. In some embodiments, a modified iRNA will have a phosphorus atom in its internucleoside backbone.

Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′ linkages, 2′-5′-linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixed salts and free acid forms are also included.

Representative US patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,625,050; 6,028,188; 6,124,445; 6,160,109; 6,169,170; 6,172,209; 6,239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423; 6,531,590; 6,534,639; 6,608,035; 6,683,167; 6,858,715; 6,867,294; 6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and U.S. Pat. RE39464, the entire contents of each of which are hereby incorporated herein by reference.

Modified RNA backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S, and CH₂ component parts.

Representative US patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439, the entire contents of each of which are hereby incorporated herein by reference.

Suitable RNA mimetics are contemplated for use in iRNAs provided herein, in which both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound in which an RNA mimetic that has been shown to have excellent hybridization properties is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of an RNA is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative US patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, the entire contents of each of which are hereby incorporated herein by reference. Additional PNA compounds suitable for use in the iRNAs of the invention are described in, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.

Some embodiments featured in the invention include RNAs with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular —CH₂—NH—CH₂—, —CH₂—N(CH₃)—O—CH₂— [known as a methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—, —CH₂—N(CH₃)—N(CH₃)—CH₂— and —N(CH₃)—CH₂—CH₂— [wherein the native phosphodiester backbone is represented as —O—P—O—CH₂—] of the above-referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above-referenced U.S. Pat. No. 5,602,240. In some embodiments, the RNAs featured herein have morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.

Modified RNAs can also contain one or more substituted sugar moieties. The iRNAs, e.g., dsRNAs, featured herein can include one of the following at the 2′-position: OH; F; O-, S-, or N-alkyl; O, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-Co-alkyl, wherein the alkyl, alkenyl and alkynyl can be substituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyl and alkynyl. Exemplary suitable modifications include O[(CH₂)_(n)O]_(m)CH₃, O(CH₂)._(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where n and m are from 1 to about 10. In other embodiments, dsRNAs include one of the following at the 2′ position: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an iRNA, or a group for improving the pharmacodynamic properties of an iRNA, and other substituents having similar properties. In some embodiments, the modification includes a 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504) i.e., an alkoxy-alkoxy group. Another exemplary modification is 2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described in examples herein below, and 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e., 2′-O—CH₂—O—CH₂—N(CH₂)₂. Further exemplary modifications include: 5′-Me-2′-F nucleotides, 5′-Me-2′-OMe nucleotides, 5′-Me-2′-deoxynucleotides, (both R and S isomers in these three families); 2′-alkoxyalkyl; and 2′-NMA (N-methylacetamide).

Other modifications include 2′-methoxy (2′-OCH₃), 2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similar modifications can also be made at other positions on the RNA of an iRNA, particularly the 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linked dsRNAs and the 5′ position of 5′ terminal nucleotide. iRNAs can also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative US patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain of which are commonly owned with the instant application. The entire contents of each of the foregoing are hereby incorporated herein by reference.

An iRNA can also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as deoxy-thymine (dT), 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl anal other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-daazaadenine and 3-deazaguanine and 3-deazaadenine. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008; those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons, 1990, these disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y S., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds featured in the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are exemplary base substitutions, even more particularly when combined with 2′-O-methoxyethyl sugar modifications.

Representative US patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Pat. Nos. 3,687,808, 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,681,941; 5,750,692; 6,015,886; 6,147,200; 6,166,197; 6,222,025; 6,235,887; 6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610; 7,427,672; and 7,495,088, the entire contents of each of which are hereby incorporated herein by reference.

The RNA of an iRNA can also be modified to include one or more locked nucleic acids (LNA). A locked nucleic acid is a nucleotide having a modified ribose moiety in which the ribose moiety comprises an extra bridge connecting the 2′ and 4′ carbons. This structure effectively “locks” the ribose in the 3′-endo structural conformation. The addition of locked nucleic acids to siRNAs has been shown to increase siRNA stability in serum, and to reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, O R. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193).

In some embodiments, the iRNA of the invention comprises one or more monomers that are UNA (unlocked nucleic acid) nucleotides. UNA is unlocked acyclic nucleic acid, wherein any of the bonds of the sugar has been removed, forming an unlocked “sugar” residue. In one example, UNA also encompasses monomer with bonds between C1′-C4′ have been removed (i.e. the covalent carbon-oxygen-carbon bond between the C1′ and C4′ carbons). In another example, the C2′-C3′ bond (i.e. the covalent carbon-carbon bond between the C2′ and C3′ carbons) of the sugar has been removed (see Nuc. Acids Symp. Series, 52, 133-134 (2008) and Fluiter et al., Mol. Biosyst., 2009, 10, 1039 hereby incorporated by reference).

The RNA of an iRNA can also be modified to include one or more bicyclic sugar moities. A “bicyclic sugar” is a furanosyl ring modified by the bridging of two atoms. A“bicyclic nucleoside” (“BNA”) is a nucleoside having a sugar moiety comprising a bridge connecting two carbon atoms of the sugar ring, thereby forming a bicyclic ring system. In certain embodiments, the bridge connects the 4′-carbon and the 2′-carbon of the sugar ring. Thus, in some embodiments an agent of the invention may include one or more locked nucleic acids (LNA). A locked nucleic acid is a nucleotide having a modified ribose moiety in which the ribose moiety comprises an extra bridge connecting the 2′ and 4′ carbons. In other words, an LNA is a nucleotide comprising a bicyclic sugar moiety comprising a 4′-CH₂—O-2′ bridge. This structure effectively “locks” the ribose in the 3′-endo structural conformation. The addition of locked nucleic acids to siRNAs has been shown to increase siRNA stability in serum, and to reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, O R. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193). Examples of bicyclic nucleosides for use in the polynucleotides of the invention include without limitation nucleosides comprising a bridge between the 4′ and the 2′ ribosyl ring atoms. In certain embodiments, the antisense polynucleotide agents of the invention include one or more bicyclic nucleosides comprising a 4′ to 2′ bridge. Examples of such 4′ to 2′ bridged bicyclic nucleosides, include but are not limited to 4′-(CH₂)—O-2′ (LNA); 4′-(CH₂)—S-2′; 4′-(CH₂)₂—O-2′ (ENA); 4′-CH(CH₃)—O-2′ (also referred to as “constrained ethyl” or “cEt”) and 4′-CH(CH₂OCH₃)—O-2′ (and analogs thereof, see, e.g., U.S. Pat. No. 7,399,845); 4′-C(CH₃)(CH₃)—O-2′ (and analogs thereof, see e.g., U.S. Pat. No. 8,278,283); 4′-CH₂—N(OCH₃)-2′ (and analogs thereof, see e.g., U.S. Pat. No. 8,278,425); 4′-CH₂—O—N(CH₃)-2′ (see, e.g., US Patent Publication No. 2004/0171570); 4′-CH₂—N(R)—O-2′, wherein R is H, C₁-C₁₂ alkyl, or a protecting group (see, e.g., U.S. Pat. No. 7,427,672); 4′-CH₂—C(H)(CH₃)-2′ (see, e.g., Chattopadhyaya et al., J. Org. Chem., 2009, 74, 118-134); and 4′-CH₂—C(═CH₂)-2′ (and analogs thereof, see, e.g., U.S. Pat. No. 8,278,426). The entire contents of each of the foregoing are hereby incorporated herein by reference.

Additional representative US patents and US patent Publications that teach the preparation of locked nucleic acid nucleotides include, but are not limited to, the following: U.S. Pat. Nos. 6,268,490; 6,525,191; 6,670,461; 6,770,748; 6,794,499; 6,998,484; 7,053,207; 7,034,133; 7,084,125; 7,399,845; 7,427,672; 7,569,686; 7,741,457; 8,022,193; 8,030,467; 8,278,425; 8,278,426; 8,278,283; US 2008/0039618; and US 2009/0012281, the entire contents of each of which are hereby incorporated herein by reference.

Any of the foregoing bicyclic nucleosides can be prepared having one or more stereochemical sugar configurations including for example α-L-ribofuranose and β-D-ribofuranose (see WO 99/14226).

The RNA of an iRNA can also be modified to include one or more constrained ethyl nucleotides. As used herein, a “constrained ethyl nucleotide” or “cEt” is a locked nucleic acid comprising a bicyclic sugar moiety comprising a 4′-CH(CH₃)—O-2′ bridge. In one embodiment, a constrained ethyl nucleotide is in the S conformation referred to herein as “S-cEt.”

An iRNA of the invention may also include one or more “conformationally restricted nucleotides” (“CRN”). CRN are nucleotide analogs with a linker connecting the C2′ and C4′ carbons of ribose or the C3 and -C5′ carbons of ribose. CRN lock the ribose ring into a stable conformation and increase the hybridization affinity to mRNA. The linker is of sufficient length to place the oxygen in an optimal position for stability and affinity resulting in less ribose ring puckering.

Representative publications that teach the preparation of certain of the above noted CRN include, but are not limited to, US Patent Publication No. 2013/0190383; and PCT publication WO 2013/036868, the entire contents of each of which are hereby incorporated herein by reference.

Representative US publications that teach the preparation of UNA include, but are not limited to, U.S. Pat. No. 8,314,227; and US Patent Publication Nos. 2013/0096289; 2013/0011922; and 2011/0313020, the entire contents of each of which are hereby incorporated herein by reference.

Potentially stabilizing modifications to the ends of RNA molecules can include N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc), N-(caproyl-4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol (Hyp-NHAc), thymidine-2′-O-deoxythymidine (ether), N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino), 2-docosanoyl-uridine-3″-phosphate, inverted base dT(idT) and others. Disclosure of this modification can be found in PCT Publication No. WO 2011/005861.

Other modifications of the nucleotides of an iRNA of the invention include a 5′ phosphate or 5′ phosphate mimic, e.g., a 5′-terminal phosphate or phosphate mimic on the antisense strand of an iRNA. Suitable phosphate mimics are disclosed in, for example US Patent Publication No. 2012/0157511, the entire contents of which are incorporated herein by reference.

A. Modified iRNAs Comprising Motifs of the Invention

In certain aspects of the invention, the double stranded RNAi agents of the invention include agents with chemical modifications as disclosed, for example, in WO2013/075035, the entire contents of each of which are incorporated herein by reference. WO2013/075035 provides motifs of three identical modifications on three consecutive nucleotides into a sense strand or antisense strand of a dsRNAi agent, particularly at or near the cleavage site. In some embodiments, the sense strand and antisense strand of the dsRNAi agent may otherwise be completely modified. The introduction of these motifs interrupts the modification pattern, if present, of the sense or antisense strand. The dsRNAi agent may be optionally conjugated with a GalNAc derivative ligand, for instance on the sense strand.

More specifically, when the sense strand and antisense strand of the double stranded RNAi agent are completely modified to have one or more motifs of three identical modifications on three consecutive nucleotides at or near the cleavage site of at least one strand of a dsRNAi agent, the gene silencing activity of the dsRNAi agent was observed.

Accordingly, the invention provides double stranded RNAi agents capable of inhibiting the expression of a target gene (i.e., IGFALS or IGF-1 gene) in vivo. The RNAi agent comprises a sense strand and an antisense strand. Each strand of the RNAi agent may be, independently, 12-30 nucleotides in length. For example, each strand may independently be 14-30 nucleotides in length, 17-30 nucleotides in length, 25-30 nucleotides in length, 27-30 nucleotides in length, 17-23 nucleotides in length, 17-21 nucleotides in length, 17-19 nucleotides in length, 19-25 nucleotides in length, 19-23 nucleotides in length, 19-21 nucleotides in length, 21-25 nucleotides in length, or 21-23 nucleotides in length.

The sense strand and antisense strand typically form a duplex double stranded RNA (“dsRNA”), also referred to herein as “dsRNAi agent.” The duplex region of a dsRNAi agent may be 12-30 nucleotide pairs in length. For example, the duplex region can be 14-30 nucleotide pairs in length, 17-30 nucleotide pairs in length, 27-30 nucleotide pairs in length, 17-23 nucleotide pairs in length, 17-21 nucleotide pairs in length, 17-19 nucleotide pairs in length, 19-25 nucleotide pairs in length, 19-23 nucleotide pairs in length, 19-21 nucleotide pairs in length, 21-25 nucleotide pairs in length, or 21-23 nucleotide pairs in length. In another example, the duplex region is selected from 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

In certain embodiments, the sense and antisense strands may be even longer. For example, in certain embodiments, the sense strand and the antisense strand are independently 25-35 nucleotides in length. In certain embodiments, each the sense and the antisense strand are independently 27-53 nucleotides in length, e.g., 27-49, 31-49, 33-49, 35-49, 37-49, and 39-49 nucleotides in length.

In certain embodiments, the dsRNAi agent may contain one or more overhang regions or capping groups at the 3′-end, 5′-end, or both ends of one or both strands. The overhang can be, independently, 1-6 nucleotides in length, for instance 2-6 nucleotides in length, 1-5 nucleotides in length, 2-5 nucleotides in length, 1-4 nucleotides in length, 2-4 nucleotides in length, 1-3 nucleotides in length, 2-3 nucleotides in length, or 1-2 nucleotides in length. In certain embodiments, at least one strand of the dsRNAi agent comprises a 3′ overhang of at least 1 nucleotide. In another embodiment, at least one strand comprises a 3′ overhang of at least 2 nucleotides, e.g., 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In other embodiments, at least one strand of the dsRNAi agent comprises a 5′ overhang of at least 1 nucleotide. In certain embodiments, at least one strand comprises a 5′ overhang of at least 2 nucleotides, e.g., 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In still other embodiments, both the 3′ and the 5′ end of one strand of the dsRNAi agent comprise an overhang of at least 1 nucleotide.

In certain embodiments, the overhang regions can include extended overhang regions as provided above. The overhangs can be the result of one strand being longer than the other, or the result of two strands of the same length being staggered. The overhang can form a mismatch with the target mRNA or it can be complementary to the gene sequences being targeted or can be another sequence. The first and second strands can also be joined, e.g., by additional bases to form a hairpin, or by other non-base linkers.

In certain embodiments, the nucleotides in the overhang region of the dsRNAi agent can each independently be a modified or unmodified nucleotide including, but no limited to 2′-sugar modified, such as, 2′-F, 2′-O-methyl, thymidine (T), 2′-O-methoxyethyl-5-methyluridine (Teo), 2′-O-methoxyethyladenosine (Aeo), 2′-O-methoxyethyl-5-methylcytidine (m5Ceo), and any combinations thereof. For example, TT can be an overhang sequence for either end on either strand. The overhang can form a mismatch with the target mRNA or it can be complementary to the gene sequences being targeted or can be another sequence.

The 5′- or 3′-overhangs at the sense strand, antisense strand, or both strands of the dsRNAi agent may be phosphorylated. In some embodiments, the overhang region(s) contains two nucleotides having a phosphorothioate between the two nucleotides, where the two nucleotides can be the same or different. In some embodiments, the overhang is present at the 3′-end of the sense strand, antisense strand, or both strands. In some embodiments, this 3′-overhang is present in the antisense strand. In some embodiments, this 3′-overhang is present in the sense strand.

The dsRNAi agent may contain only a single overhang, which can strengthen the interference activity of the RNAi, without affecting its overall stability. For example, the single-stranded overhang may be located at the 3′-end of the sense strand or, alternatively, at the 3′-end of the antisense strand. The RNAi may also have a blunt end, located at the 5′-end of the antisense strand (or the 3′-end of the sense strand) or vice versa. Generally, the antisense strand of the dsRNAi agent has a nucleotide overhang at the 3′-end, and the 5′-end is blunt. While not wishing to be bound by theory, the asymmetric blunt end at the 5′-end of the antisense strand and 3′-end overhang of the antisense strand favor the guide strand loading into RISC process.

In certain embodiments, the dsRNAi agent is a double ended bluntmer of 19 nucleotides in length, wherein the sense strand contains at least one motif of three 2′-F modifications on three consecutive nucleotides at positions 7, 8, 9 from the 5′end. The antisense strand contains at least one motif of three 2′-O-methyl modifications on three consecutive nucleotides at positions 11, 12, 13 from the 5′end.

In other embodiments, the dsRNAi agent is a double ended bluntmer of 20 nucleotides in length, wherein the sense strand contains at least one motif of three 2′-F modifications on three consecutive nucleotides at positions 8, 9, 10 from the 5′end. The antisense strand contains at least one motif of three 2′-O-methyl modifications on three consecutive nucleotides at positions 11, 12, 13 from the 5′end.

In yet other embodiments, the dsRNAi agent is a double ended bluntmer of 21 nucleotides in length, wherein the sense strand contains at least one motif of three 2′-F modifications on three consecutive nucleotides at positions 9, 10, 11 from the 5′end. The antisense strand contains at least one motif of three 2′-O-methyl modifications on three consecutive nucleotides at positions 11, 12, 13 from the 5′end.

In certain embodiments, the dsRNAi agent comprises a 21 nucleotide sense strand and a 23 nucleotide antisense strand, wherein the sense strand contains at least one motif of three 2′-F modifications on three consecutive nucleotides at positions 9, 10, 11 from the 5′end; the antisense strand contains at least one motif of three 2′-O-methyl modifications on three consecutive nucleotides at positions 11, 12, 13 from the 5′end, wherein one end of the RNAi agent is blunt, while the other end comprises a 2 nucleotide overhang. Preferably, the 2 nucleotide overhang is at the 3′-end of the antisense strand.

When the 2 nucleotide overhang is at the 3′-end of the antisense strand, there may be two phosphorothioate internucleotide linkages between the terminal three nucleotides, wherein two of the three nucleotides are the overhang nucleotides, and the third nucleotide is a paired nucleotide next to the overhang nucleotide. In one embodiment, the RNAi agent additionally has two phosphorothioate internucleotide linkages between the terminal three nucleotides at both the 5′-end of the sense strand and at the 5′-end of the antisense strand. In certain embodiments, every nucleotide in the sense strand and the antisense strand of the dsRNAi agent, including the nucleotides that are part of the motifs are modified nucleotides. In certain embodiments each residue is independently modified with a 2′-O-methyl or 3′-fluoro, e.g., in an alternating motif Optionally, the dsRNAi agent further comprises a ligand (preferably GalNAc₃).

In certain embodiments, the dsRNAi agent comprises a sense and an antisense strand, wherein the sense strand is 25-30 nucleotide residues in length, wherein starting from the 5′ terminal nucleotide (position 1) positions 1 to 23 of the first strand comprise at least 8 ribonucleotides; the antisense strand is 36-66 nucleotide residues in length and, starting from the 3′ terminal nucleotide, comprises at least 8 ribonucleotides in the positions paired with positions 1-23 of sense strand to form a duplex; wherein at least the 3′ terminal nucleotide of antisense strand is unpaired with sense strand, and up to 6 consecutive 3′ terminal nucleotides are unpaired with sense strand, thereby forming a 3′ single stranded overhang of 1-6 nucleotides; wherein the 5′ terminus of antisense strand comprises from 10-30 consecutive nucleotides which are unpaired with sense strand, thereby forming a 10-30 nucleotide single stranded 5′ overhang; wherein at least the sense strand 5′ terminal and 3′ terminal nucleotides are base paired with nucleotides of antisense strand when sense and antisense strands are aligned for maximum complementarity, thereby forming a substantially duplexed region between sense and antisense strands; and antisense strand is sufficiently complementary to a target RNA along at least 19 ribonucleotides of antisense strand length to reduce target gene expression when the double stranded nucleic acid is introduced into a mammalian cell; and wherein the sense strand contains at least one motif of three 2′-F modifications on three consecutive nucleotides, where at least one of the motifs occurs at or near the cleavage site. The antisense strand contains at least one motif of three 2′-O-methyl modifications on three consecutive nucleotides at or near the cleavage site.

In certain embodiments, the dsRNAi agent comprises sense and antisense strands, wherein the dsRNAi agent comprises a first strand having a length which is at least 25 and at most 29 nucleotides and a second strand having a length which is at most 30 nucleotides with at least one motif of three 2′-O-methyl modifications on three consecutive nucleotides at position 11, 12, 13 from the 5′ end; wherein the 3′ end of the first strand and the 5′ end of the second strand form a blunt end and the second strand is 1-4 nucleotides longer at its 3′ end than the first strand, wherein the duplex region region which is at least 25 nucleotides in length, and the second strand is sufficiently complementary to a target mRNA along at least 19 nucleotide of the second strand length to reduce target gene expression when the RNAi agent is introduced into a mammalian cell, and wherein Dicer cleavage of the dsRNAi agent preferentially results in an siRNA comprising the 3′-end of the second strand, thereby reducing expression of the target gene in the mammal. Optionally, the dsRNAi agent further comprises a ligand.

In certain embodiments, the sense strand of the dsRNAi agent contains at least one motif of three identical modifications on three consecutive nucleotides, where one of the motifs occurs at the cleavage site in the sense strand.

In certain embodiments, the antisense strand of the dsRNAi agent can also contain at least one motif of three identical modifications on three consecutive nucleotides, where one of the motifs occurs at or near the cleavage site in the antisense strand.

For a dsRNAi agent having a duplex region of 17-23 nucleotides in length, the cleavage site of the antisense strand is typically around the 10, 11, and 12 positions from the 5′-end. Thus the motifs of three identical modifications may occur at the 9, 10, 11 positions; the 10, 11, 12 positions; the 11, 12, 13 positions; the 12, 13, 14 positions; or the 13, 14, 15 positions of the antisense strand, the count starting from the first nucleotide from the 5′-end of the antisense strand, or, the count starting from the first paired nucleotide within the duplex region from the 5′-end of the antisense strand. The cleavage site in the antisense strand may also change according to the length of the duplex region of the dsRNAi agent from the 5′-end.

The sense strand of the dsRNAi agent may contain at least one motif of three identical modifications on three consecutive nucleotides at the cleavage site of the strand; and the antisense strand may have at least one motif of three identical modifications on three consecutive nucleotides at or near the cleavage site of the strand. When the sense strand and the antisense strand form a dsRNA duplex, the sense strand and the antisense strand can be so aligned that one motif of the three nucleotides on the sense strand and one motif of the three nucleotides on the antisense strand have at least one nucleotide overlap, i.e., at least one of the three nucleotides of the motif in the sense strand forms a base pair with at least one of the three nucleotides of the motif in the antisense strand. Alternatively, at least two nucleotides may overlap, or all three nucleotides may overlap.

In some embodiments, the sense strand of the dsRNAi agent may contain more than one motif of three identical modifications on three consecutive nucleotides. The first motif may occur at or near the cleavage site of the strand and the other motifs may be a wing modification. The term “wing modification” herein refers to a motif occurring at another portion of the strand that is separated from the motif at or near the cleavage site of the same strand. The wing modification is either adjacent to the first motif or is separated by at least one or more nucleotides. When the motifs are immediately adjacent to each other then the chemistries of the motifs are distinct from each other, and when the motifs are separated by one or more nucleotide than the chemistries can be the same or different. Two or more wing modifications may be present. For instance, when two wing modifications are present, each wing modification may occur at one end relative to the first motif which is at or near cleavage site or on either side of the lead motif.

Like the sense strand, the antisense strand of the dsRNAi agent may contain more than one motif of three identical modifications on three consecutive nucleotides, with at least one of the motifs occurring at or near the cleavage site of the strand. This antisense strand may also contain one or more wing modifications in an alignment similar to the wing modifications that may be present on the sense strand.

In some embodiments, the wing modification on the sense strand or antisense strand of the dsRNAi agent typically does not include the first one or two terminal nucleotides at the 3′-end, 5′-end, or both ends of the strand.

In other embodiments, the wing modification on the sense strand or antisense strand of the dsRNAi agent typically does not include the first one or two paired nucleotides within the duplex region at the 3′-end, 5′-end, or both ends of the strand.

When the sense strand and the antisense strand of the dsRNAi agent each contain at least one wing modification, the wing modifications may fall on the same end of the duplex region, and have an overlap of one, two, or three nucleotides.

When the sense strand and the antisense strand of the dsRNAi agent each contain at least two wing modifications, the sense strand and the antisense strand can be so aligned that two modifications each from one strand fall on one end of the duplex region, having an overlap of one, two, or three nucleotides; two modifications each from one strand fall on the other end of the duplex region, having an overlap of one, two or three nucleotides; two modifications one strand fall on each side of the lead motif, having an overlap of one, two or three nucleotides in the duplex region.

In some embodiments, every nucleotide in the sense strand and antisense strand of the dsRNAi agent, including the nucleotides that are part of the motifs, may be modified. Each nucleotide may be modified with the same or different modification which can include one or more alteration of one or both of the non-linking phosphate oxygens or of one or more of the linking phosphate oxygens; alteration of a constituent of the ribose sugar, e.g., of the 2′-hydroxyl on the ribose sugar; wholesale replacement of the phosphate moiety with “dephospho” linkers; modification or replacement of a naturally occurring base; and replacement or modification of the ribose-phosphate backbone.

As nucleic acids are polymers of subunits, many of the modifications occur at a position which is repeated within a nucleic acid, e.g., a modification of a base, or a phosphate moiety, or a non-linking O of a phosphate moiety. In some cases the modification will occur at all of the subject positions in the nucleic acid but in many cases it will not. By way of example, a modification may only occur at a 3′- or 5′-terminal position, may only occur in a terminal region, e.g., at a position on a terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of a strand. A modification may occur in a double strand region, a single strand region, or in both. A modification may occur only in the double strand region of a dsRNAi agent or may only occur in a single strand region of a dsRNAi agent. For example, a phosphorothioate modification at a non-linking O position may only occur at one or both ends, may only occur in a terminal region, e.g., at a position on a terminal nucleotide, or in the last 2, 3, 4, 5, or 10 nucleotides of a strand, or may occur in double strand and single strand regions, particularly at the ends. The 5′-end or ends can be phosphorylated.

It may be possible, e.g., to enhance stability, to include particular bases in overhangs, or to include modified nucleotides or nucleotide surrogates, in single strand overhangs, e.g., in a 5′- or 3′-overhang, or in both. For example, it can be desirable to include purine nucleotides in overhangs. In some embodiments all or some of the bases in a 3′- or 5′-overhang may be modified, e.g., with a modification described herein. Modifications can include, e.g., the use of modifications at the 2′ position of the ribose sugar with modifications that are known in the art, e.g., the use of deoxyribonucleotides, 2′-deoxy-2′-fluoro (2′-F) or 2′-O-methyl modified instead of the ribosugar of the nucleobase, and modifications in the phosphate group, e.g., phosphorothioate modifications. Overhangs need not be homologous with the target sequence.

In some embodiments, each residue of the sense strand and antisense strand is independently modified with LNA, CRN, cET, UNA, HNA, CeNA, 2′-methoxyethyl, 2′-O-methyl, 2′-O-allyl, 2′-C-allyl, 2′-deoxy, 2′-hydroxyl, or 2′-fluoro. The strands can contain more than one modification. In one embodiment, each residue of the sense strand and antisense strand is independently modified with 2′-O-methyl or 2′-fluoro.

At least two different modifications are typically present on the sense strand and antisense strand. Those two modifications may be the 2′-O-methyl or 2′-fluoro modifications, or others.

In certain embodiments, the N_(a) or N_(b) comprise modifications of an alternating pattern. The term “alternating motif” as used herein refers to a motif having one or more modifications, each modification occurring on alternating nucleotides of one strand. The alternating nucleotide may refer to one per every other nucleotide or one per every three nucleotides, or a similar pattern. For example, if A, B and C each represent one type of modification to the nucleotide, the alternating motif can be “ABABABABABAB . . . ,” “AABBAABBAABB . . . ,” “AABAABAABAAB . . . ,” “AAABAAABAAAB . . . ,” “AAABBBAAABBB . . . ,” or “ABCABCABCABC . . . ,” etc.

The type of modifications contained in the alternating motif may be the same or different. For example, if A, B, C, D each represent one type of modification on the nucleotide, the alternating pattern, i.e., modifications on every other nucleotide, may be the same, but each of the sense strand or antisense strand can be selected from several possibilities of modifications within the alternating motif such as “ABABAB . . . ”, “ACACAC . . . ” “BDBDBD . . . ” or “CDCDCD . . . ,” etc.

In some embodiments, the dsRNAi agent of the invention comprises the modification pattern for the alternating motif on the sense strand relative to the modification pattern for the alternating motif on the antisense strand is shifted. The shift may be such that the modified group of nucleotides of the sense strand corresponds to a differently modified group of nucleotides of the antisense strand and vice versa. For example, the sense strand when paired with the antisense strand in the dsRNA duplex, the alternating motif in the sense strand may start with “ABABAB” from 5′ to 3′ of the strand and the alternating motif in the antisense strand may start with “BABABA” from 5′ to 3′ of the strand within the duplex region. As another example, the alternating motif in the sense strand may start with “AABBAABB” from 5′ to 3′ of the strand and the alternating motif in the antisenese strand may start with “BBAABBAA” from 5′ to 3′ of the strand within the duplex region, so that there is a complete or partial shift of the modification patterns between the sense strand and the antisense strand.

In some embodiments, the dsRNAi agent comprises the pattern of the alternating motif of 2′-O-methyl modification and 2′-F modification on the sense strand initially has a shift relative to the pattern of the alternating motif of 2′-O-methyl modification and 2′-F modification on the antisense strand initially, i.e., the 2′-O-methyl modified nucleotide on the sense strand base pairs with a 2′-F modified nucleotide on the antisense strand and vice versa. The 1 position of the sense strand may start with the 2′-F modification, and the 1 position of the antisense strand may start with the 2′-O-methyl modification.

The introduction of one or more motifs of three identical modifications on three consecutive nucleotides to the sense strand or antisense strand interrupts the initial modification pattern present in the sense strand or antisense strand. This interruption of the modification pattern of the sense or antisense strand by introducing one or more motifs of three identical modifications on three consecutive nucleotides to the sense or antisense strand may enhance the gene silencing activity against the target gene.

In some embodiments, when the motif of three identical modifications on three consecutive nucleotides is introduced to any of the strands, the modification of the nucleotide next to the motif is a different modification than the modification of the motif. For example, the portion of the sequence containing the motif is “ . . . N_(a)YYYN_(b) . . . ,” where “Y” represents the modification of the motif of three identical modifications on three consecutive nucleotide, and “N_(a)” and “N_(b)” represent a modification to the nucleotide next to the motif “YYY” that is different than the modification of Y, and where N_(a) and N_(b) can be the same or different modifications. Alternatively, N_(a) or N_(b) may be present or absent when there is a wing modification present.

The iRNA may further comprise at least one phosphorothioate or methylphosphonate internucleotide linkage. The phosphorothioate or methylphosphonate internucleotide linkage modification may occur on any nucleotide of the sense strand, antisense strand, or both strands in any position of the strand. For instance, the internucleotide linkage modification may occur on every nucleotide on the sense strand or antisense strand; each internucleotide linkage modification may occur in an alternating pattern on the sense strand or antisense strand; or the sense strand or antisense strand may contain both internucleotide linkage modifications in an alternating pattern. The alternating pattern of the internucleotide linkage modification on the sense strand may be the same or different from the antisense strand, and the alternating pattern of the internucleotide linkage modification on the sense strand may have a shift relative to the alternating pattern of the internucleotide linkage modification on the antisense strand. In one embodiment, a double-stranded RNAi agent comprises 6-8phosphorothioate internucleotide linkages. In some embodiments, the antisense strand comprises two phosphorothioate internucleotide linkages at the 5′-end and two phosphorothioate internucleotide linkages at the 3′-end, and the sense strand comprises at least two phosphorothioate internucleotide linkages at either the 5′-end or the 3′-end.

In some embodiments, the dsRNAi agent comprises a phosphorothioate or methylphosphonate internucleotide linkage modification in the overhang region. For example, the overhang region may contain two nucleotides having a phosphorothioate or methylphosphonate internucleotide linkage between the two nucleotides. Internucleotide linkage modifications also may be made to link the overhang nucleotides with the terminal paired nucleotides within the duplex region. For example, at least 2, 3, 4, or all the overhang nucleotides may be linked through phosphorothioate or methylphosphonate internucleotide linkage, and optionally, there may be additional phosphorothioate or methylphosphonate internucleotide linkages linking the overhang nucleotide with a paired nucleotide that is next to the overhang nucleotide. For instance, there may be at least two phosphorothioate internucleotide linkages between the terminal three nucleotides, in which two of the three nucleotides are overhang nucleotides, and the third is a paired nucleotide next to the overhang nucleotide. These terminal three nucleotides may be at the 3′-end of the antisense strand, the 3′-end of the sense strand, the 5′-end of the antisense strand, or the 5′end of the antisense strand.

In some embodiments, the 2-nucleotide overhang is at the 3′-end of the antisense strand, and there are two phosphorothioate internucleotide linkages between the terminal three nucleotides, wherein two of the three nucleotides are the overhang nucleotides, and the third nucleotide is a paired nucleotide next to the overhang nucleotide. Optionally, the dsRNAi agent may additionally have two phosphorothioate internucleotide linkages between the terminal three nucleotides at both the 5′-end of the sense strand and at the 5′-end of the antisense strand.

In one embodiment, the dsRNAi agent comprises mismatch(es) with the target, within the duplex, or combinations thereof. The mistmatch may occur in the overhang region or the duplex region. The base pair may be ranked on the basis of their propensity to promote dissociation or melting (e.g., on the free energy of association or dissociation of a particular pairing, the simplest approach is to examine the pairs on an individual pair basis, though next neighbor or similar analysis can also be used). In terms of promoting dissociation: A:U is preferred over G:C; G:U is preferred over G:C; and I:C is preferred over G:C (I=inosine). Mismatches, e.g., non-canonical or other than canonical pairings (as described elsewhere herein) are preferred over canonical (A:T, A:U, G:C) pairings; and pairings which include a universal base are preferred over canonical pairings.

In certain embodiments, the dsRNAi agent comprises at least one of the first 1, 2, 3, 4, or 5 base pairs within the duplex regions from the 5′-end of the antisense strand independently selected from the group of: A:U, G:U, I:C, and mismatched pairs, e.g., non-canonical or other than canonical pairings or pairings which include a universal base, to promote the dissociation of the antisense strand at the 5′-end of the duplex.

In certain embodiments, the nucleotide at the 1 position within the duplex region from the 5′-end in the antisense strand is selected from A, dA, dU, U, and dT. Alternatively, at least one of the first 1, 2, or 3 base pair within the duplex region from the 5′-end of the antisense strand is an AU base pair. For example, the first base pair within the duplex region from the 5′-end of the antisense strand is an AU base pair.

In other embodiments, the nucleotide at the 3′-end of the sense strand is deoxy-thymine (dT) or the nucleotide at the 3′-end of the antisense strand is deoxy-thymine (dT). For example, there is a short sequence of deoxy-thymine nucleotides, for example, two dT nucleotides on the 3′-end of the sense, antisense strand, or both strands.

In certain embodiments, the sense strand sequence may be represented by formula (I):

5′ n_(p)-N_(a)-(X X X )i-N_(b)-Y Y Y -N_(b)-(Z Z Z )_(j)-N_(a)-n_(q) 3′ (I)

wherein:

i and j are each independently 0 or 1;

p and q are each independently 0-6;

each N_(a) independently represents an oligonucleotide sequence comprising 0-25 modified nucleotides, each sequence comprising at least two differently modified nucleotides;

each N_(b) independently represents an oligonucleotide sequence comprising 0-10 modified nucleotides;

each n_(p) and n_(q) independently represent an overhang nucleotide;

wherein Nb and Y do not have the same modification; and

XXX, YYY, and ZZZ each independently represent one motif of three identical modifications on three consecutive nucleotides. Preferably YYY is all 2′-F modified nucleotides.

In some embodiments, the N_(a) or N_(b) comprises modifications of alternating pattern.

In some embodiments, the YYY motif occurs at or near the cleavage site of the sense strand. For example, when the dsRNAi agent has a duplex region of 17-23 nucleotides in length, the YYY motif can occur at or the vicinity of the cleavage site (e.g.: can occur at positions 6, 7, 8; 7, 8, 9; 8, 9, 10; 9, 10, 11; 10, 11, 12; or 11, 12, 13) of the sense strand, the count starting from the first nucleotide, from the 5′-end; or optionally, the count starting at the first paired nucleotide within the duplex region, from the 5′-end.

In one embodiment, i is 1 and j is 0, or i is 0 and j is 1, or both i and j are 1. The sense strand can therefore be represented by the following formulas:

5′ n_(p)-N_(a)-YYY-N_(b)-ZZZ-N_(a)-n_(q) 3′ (Ib); 5′ n_(p)-N_(a)-XXX-N_(b)-YYY-N_(a)-n_(q) 3′ (Ic); or 5′ n_(p)-N_(a)-XXX-N_(b)-YYY-N_(b)-ZZZ-N_(a)-n_(q) 3′ (Id).

When the sense strand is represented by formula (Ib), N_(b) represents an oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. Each N_(a) independently can represent an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the sense strand is represented as formula (Ic), N_(b) represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. Each N_(a) can independently represent an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the sense strand is represented as formula (Id), each N_(b) independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. Preferably, N_(b) is 0, 1, 2, 3, 4, 5, or 6 Each N_(a) can independently represent an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

Each of X, Y and Z may be the same or different from each other.

In other embodiments, i is 0 and j is 0, and the sense strand may be represented by the formula:

5′ n_(p)-N_(a)-YYY- N_(a)-n_(q) 3′ (Ia).

When the sense strand is represented by formula (Ia), each N_(a) independently can represent an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

In one embodiment, the antisense strand sequence of the RNAi may be represented by formula (II):

5′ n_(q′)-N_(a)′-(Z′Z′Z′)_(k)-N_(b)′-Y′Y′Y′-N_(b)′-(X′X′X′)_(l)-N′_(a)- n_(p)′ 3′ (II)

wherein:

k and l are each independently 0 or 1;

p′ and q′ are each independently 0-6;

each N_(a)′ independently represents an oligonucleotide sequence comprising 0-25 modified nucleotides, each sequence comprising at least two differently modified nucleotides;

each N_(b)′ independently represents an oligonucleotide sequence comprising 0-10 modified nucleotides;

each n_(p)′ and n_(q)′ independently represent an overhang nucleotide;

wherein N_(b)′ and Y′ do not have the same modification; and

X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently represent one motif of three identical modifications on three consecutive nucleotides.

In some embodiments, the N_(a)′ or N_(b)′ comprises modifications of alternating pattern.

The Y′Y′Y′ motif occurs at or near the cleavage site of the antisense strand. For example, when the dsRNAi agent has a duplex region of 17-23 nucleotides in length, the Y′Y′Y′ motif can occur at positions 9, 10, 11; 10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14, 15 of the antisense strand, with the count starting from the first nucleotide, from the 5′-end; or optionally, the count starting at the first paired nucleotide within the duplex region, from the 5′-end. Preferably, the Y′Y′Y′ motif occurs at positions 11, 12, 13.

In certain embodiments, Y′Y′Y′ motif is all 2′-OMe modified nucleotides.

In certain embodiments, k is 1 and l is 0, or k is 0 and l is 1, or both k and l are 1.

The antisense strand can therefore be represented by the following formulas:

5′ n_(q′)-N_(a)′-Z′Z′Z′-N_(b)′-Y′Y′Y′-N_(a)′-n_(p′) 3′ (IIb); 5′ n_(q′)-N_(a)′-Y′Y′Y′-N_(b)′-X′X′X′-n_(p′) 3′ (IIc); or 5′ n_(q′)-N_(a)′- Z′Z′Z′-N_(b)′-Y′Y′Y′-N_(b)′- X′X′X′-N_(a)′-n_(p′) 3′ (IId).

When the antisense strand is represented by formula (IIb), N_(b)′ represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. Each N_(a)′ independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the antisense strand is represented as formula (IIc), N_(b)′ represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. Each N_(a)′ independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the antisense strand is represented as formula (IId), each N_(b)′ independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. Each N_(a)′ independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides. Preferably, N_(b) is 0, 1, 2, 3, 4, 5, or 6.

In other embodiments, k is 0 and 1 is 0 and the antisense strand may be represented by the formula:

5′ n_(p′)-N_(a′)-Y′Y′Y′- N_(a′)-n_(q′) 3′ (Ia).

When the antisense strand is represented as formula (IIa), each N_(a)′ independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides. Each of X′, Y′ and Z′ may be the same or different from each other.

Each nucleotide of the sense strand and antisense strand may be independently modified with LNA, CRN, UNA, cEt, HNA, CeNA, 2′-methoxyethyl, 2′-O-methyl, 2′-O-allyl, 2′-C-allyl, 2′-hydroxyl, or 2′-fluoro. For example, each nucleotide of the sense strand and antisense strand is independently modified with 2′-O-methyl or 2′-fluoro. Each X, Y, Z, X′, Y′, and Z′, in particular, may represent a 2′-O-methyl modification or a 2′-fluoro modification.

In some embodiments, the sense strand of the dsRNAi agent may contain YYY motif occurring at 9, 10, and 11 positions of the strand when the duplex region is 21 nt, the count starting from the first nucleotide from the 5′-end, or optionally, the count starting at the first paired nucleotide within the duplex region, from the 5′-end; and Y represents 2′-F modification. The sense strand may additionally contain XXX motif or ZZZ motifs as wing modifications at the opposite end of the duplex region; and XXX and ZZZ each independently represents a 2′-OMe modification or 2′-F modification.

In some embodiments the antisense strand may contain Y′Y′Y′ motif occurring at positions 11, 12, 13 of the strand, the count starting from the first nucleotide from the 5′-end, or optionally, the count starting at the first paired nucleotide within the duplex region, from the 5′-end; and Y′ represents 2′-O-methyl modification. The antisense strand may additionally contain X′X′X′ motif or Z′Z′Z′ motifs as wing modifications at the opposite end of the duplex region; and X′X′X′ and Z′Z′Z′ each independently represents a 2′-OMe modification or 2′-F modification.

The sense strand represented by any one of the above formulas (Ia), (Ib), (Ic), and (Id) forms a duplex with a antisense strand being represented by any one of formulas (IIa), (IIb), (IIc), and (IId), respectively.

Accordingly, the dsRNAi agents for use in the methods of the invention may comprise a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, the iRNA duplex represented b formula (III):

sense: 5′ n_(p) -N_(a)-(X X X)_(i)-N_(b)- Y Y Y -N_(b) -(Z Z Z)_(j)-N_(a)-n_(q) 3′ antisense: 3′ n_(p)′-N_(a)′-(X′X′X′)_(k)-N_(b)′-Y′Y′Y′-N_(b)′-(Z′Z′Z′)_(l)-N_(a)′- n_(q)′ 5′ (III)

wherein:

i, j, k, and 1 are each independently 0 or 1;

p, p′, q, and q′ are each independently 0-6;

each N_(a) and N_(a)′ independently represents an oligonucleotide sequence comprising 0-25 modified nucleotides, each sequence comprising at least two differently modified nucleotides; each N_(b) and N_(b)′ independently represents an oligonucleotide sequence comprising 0-10 modified nucleotides;

wherein each n_(p)′, n_(p), n_(q)′, and n_(q), each of which may or may not be present, independently represents an overhang nucleotide; and

XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently represent one motif of three identical modifications on three consecutive nucleotides.

In one embodiment, i is 0 and j is 0; or i is 1 and j is 0; or i is 0 and j is 1; or both i and j are 0; or both i and j are 1. In another embodiment, k is 0 and l is 0; or k is 1 and l is 0; k is 0 and l is 1; or both k and l are 0; or both k and l are 1.

Exemplary combinations of the sense strand and antisense strand forming an iRNA duplex include the formulas below:

(IIIa) 5′ n_(p)-N_(a)-Y Y Y-N_(a)-n_(q) 3′ 3′ n_(p)'-N_(a')-Y'Y'Y'-N_(a')n_(q') 5′ (IIIb) 5′ n_(p)-N_(a)-Y Y Y-N_(b)-Z Z Z-N_(a)-n_(q) 3′ 3′ n_(p')-N_(a')-Y'Y'Y'-N_(b')-Z'Z'Z'-N_(a')n_(q') 5′ (IIIc) 5′ n_(p)-N_(a)-X X X-N_(b)-Y Y Y-N_(a)-n_(q) 3′  3′ n_(p')-N_(a')-X'X'X'-N_(b')-Y'Y'Y'-N_(a')-n_(q') 5′ (IIId) 5′ n_(p)-N_(a)-X X X-N_(b)-Y Y Y-N_(b)-Z Z Z-N_(a)-n_(q) 3′ 3′ n_(p')-N_(a')-X'X'X'-N_(b')-Y'Y'Y'-N_(b')-Z'Z'Z'- N_(a)-n_(q') 5′

When the dsRNAi agent is represented by formula (IIIa), each N_(a) independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the dsRNAi agent is represented by formula (IIIb), each N_(b) independently represents an oligonucleotide sequence comprising 1-10, 1-7, 1-5, or 1-4 modified nucleotides. Each N_(a) independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the dsRNAi agent is represented as formula (IIc), each N_(b), N_(b)′ independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. Each N_(a) independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the dsRNAi agent is represented as formula (IIId), each N_(b), N_(b)′ independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. Each N_(a), N_(a)′ independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides. Each of N_(a), N_(a)′, N_(b), and N_(b)′ independently comprises modifications of alternating pattern.

Each of X, Y, and Z in formulas (III), (IIIa), (IIIb), (IIIc), and (IIId) may be the same or different from each other.

When the dsRNAi agent is represented by formula (III), (IIIa), (IIIb), (IIIc), and (IIId), at least one of the Y nucleotides may form a base pair with one of the Y′ nucleotides. Alternatively, at least two of the Y nucleotides form base pairs with the corresponding Y′ nucleotides; or all three of the Y nucleotides all form base pairs with the corresponding Y′ nucleotides.

When the dsRNAi agent is represented by formula (IIIb) or (IIId), at least one of the Z nucleotides may form a base pair with one of the Z′ nucleotides. Alternatively, at least two of the Z nucleotides form base pairs with the corresponding Z′ nucleotides; or all three of the Z nucleotides all form base pairs with the corresponding Z′ nucleotides.

When the dsRNAi agent is represented as formula (IIIc) or (IIId), at least one of the X nucleotides may form a base pair with one of the X′ nucleotides. Alternatively, at least two of the X nucleotides form base pairs with the corresponding X′ nucleotides; or all three of the X nucleotides all form base pairs with the corresponding X′ nucleotides.

In certain embodiments, the modification on the Y nucleotide is different than the modification on the Y′ nucleotide, the modification on the Z nucleotide is different than the modification on the Z′ nucleotide, and/or the modification on the X nucleotide is different than the modification on the X′ nucleotide.

In certain embodiments, when the dsRNAi agent is represented by formula (IIId), the N_(a) modifications are 2′-O-methyl or 2′-fluoro modifications. In other embodiments, when the RNAi agent is represented by formula (IIId), the N_(a) modifications are 2′-O-methyl or 2′-fluoro modifications and n_(p)′>0 and at least one n_(p)′ is linked to a neighboring nucleotide a via phosphorothioate linkage. In yet other embodiments, when the RNAi agent is represented by formula (IIId), the N_(a) modifications are 2′-O-methyl or 2′-fluoro modifications, n_(p)′>0 and at least one n_(p)′ is linked to a neighboring nucleotide via phosphorothioate linkage, and the sense strand is conjugated to one or more GalNAc derivatives attached through a monovalent, a bivalent or a trivalent branched linker (described below).

In other embodiments, when the RNAi agent is represented by formula (IIId), the N_(a) modifications are 2′-O-methyl or 2′-fluoro modifications, n_(p)′>0 and at least one n_(p)′ is linked to a neighboring nucleotide via phosphorothioate linkage, the sense strand comprises at least one phosphorothioate linkage, and the sense strand is conjugated to one or more GalNAc derivatives attached through a monovalent, a bivalent or a trivalent branched linker.

In some embodiments, when the dsRNAi agent is represented by formula (IIIa), the N_(a) modifications are 2′-O-methyl or 2′-fluoro modifications, n_(p)′>0 and at least one n_(p)′ is linked to a neighboring nucleotide via phosphorothioate linkage, the sense strand comprises at least one phosphorothioate linkage, and the sense strand is conjugated to one or more GalNAc derivatives attached through a monovalent, a bivalent or a trivalent branched linker.

In some embodiments, the dsRNAi agent is a multimer containing at least two duplexes represented by formula (III), (IIIa), (IIIb), (IIIc), and (IIId), wherein the duplexes are connected by a linker. The linker can be cleavable or non-cleavable. Optionally, the multimer further comprises a ligand. Each of the duplexes can target the same gene or two different genes; or each of the duplexes can target same gene at two different target sites.

In some embodiments, the dsRNAi agent is a multimer containing three, four, five, six, or more duplexes represented by formula (III), (IIIa), (IIIb), (IIIc), and (IIId), wherein the duplexes are connected by a linker. The linker can be cleavable or non-cleavable. Optionally, the multimer further comprises a ligand. Each of the duplexes can target the same gene or two different genes; or each of the duplexes can target same gene at two different target sites.

In one embodiment, two dsRNAi agents represented by at least one of formulas (III), (IIIa), (IIIb), (IIIc), and (IIId) are linked to each other at the 5′ end, and one or both of the 3′ ends, and are optionally conjugated to to a ligand. Each of the agents can target the same gene or two different genes; or each of the agents can target same gene at two different target sites.

Various publications describe multimeric iRNAs that can be used in the methods of the invention. Such publications include U.S. Pat. No. 7,858,769, WO2007/091269, WO2010/141511, WO2007/117686, WO2009/014887, and WO2011/031520 the entire contents of each of which are hereby incorporated herein by reference.

As described in more detail below, the iRNA that contains conjugations of one or more carbohydrate moieties to an iRNA can optimize one or more properties of the iRNA. In many cases, the carbohydrate moiety will be attached to a modified subunit of the iRNA. For example, the ribose sugar of one or more ribonucleotide subunits of a iRNA can be replaced with another moiety, e.g., a non-carbohydrate (preferably cyclic) carrier to which is attached a carbohydrate ligand. A ribonucleotide subunit in which the ribose sugar of the subunit has been so replaced is referred to herein as a ribose replacement modification subunit (RRMS). A cyclic carrier may be a carbocyclic ring system, i.e., all ring atoms are carbon atoms, or a heterocyclic ring system, i.e., one or more ring atoms may be a heteroatom, e.g., nitrogen, oxygen, sulfur. The cyclic carrier may be a monocyclic ring system, or may contain two or more rings, e.g. fused rings. The cyclic carrier may be a fully saturated ring system, or it may contain one or more double bonds.

The ligand may be attached to the polynucleotide via a carrier. The carriers include (i) at least one “backbone attachment point,” preferably two “backbone attachment points” and (ii) at least one “tethering attachment point.” A “backbone attachment point” as used herein refers to a functional group, e.g. a hydroxyl group, or generally, a bond available for, and that is suitable for incorporation of the carrier into the backbone, e.g., the phosphate, or modified phosphate, e.g., sulfur containing, backbone, of a ribonucleic acid. A “tethering attachment point” (TAP) in some embodiments refers to a constituent ring atom of the cyclic carrier, e.g., a carbon atom or a heteroatom (distinct from an atom which provides a backbone attachment point), that connects a selected moiety. The moiety can be, e.g., a carbohydrate, e.g. monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide. Optionally, the selected moiety is connected by an intervening tether to the cyclic carrier. Thus, the cyclic carrier will often include a functional group, e.g., an amino group, or generally, provide a bond, that is suitable for incorporation or tethering of another chemical entity, e.g., a ligand to the constituent ring.

The iRNA may be conjugated to a ligand via a carrier, wherein the carrier can be cyclic group or acyclic group; preferably, the cyclic group is selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl, and decalin; preferably, the acyclic group is a serinol backbone or diethanolamine backbone.

In certain embodiments, the iRNA for use in the methods of the invention for inhibiting the expression of an IGFALS gene is an agent selected from the agents listed in any one of Tables 3, 5, 6, 8, 12, and 14. These agents may further comprise a ligand. These agents may further comprise a ligand.

In certain embodiments, the iRNA for use in the methods of the invention for inhibiting the expression of an IGF-1 gene is an agent selected from the agents listed in any one of Tables 9, 11, 15, 17, 18, and 20. These agents may further comprise a ligand.

III. iRNAs Conjugated to Ligands

Another modification of the RNA of an iRNA of the invention involves chemically linking to the iRNA one or more ligands, moieties or conjugates that enhance the activity, cellular distribution, or cellular uptake of the iRNA e.g., into a cell. For example, the ligand can be attached to the sense strand, antisense strand or both strands, at the 3′-end, 5′-end or both ends. For instance, the ligand may be conjugated to the sense strand. In preferred embodiments, the ligand is conjugated to the 3′-end of the sense strand. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86: 6553-6556), cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4:1053-1060). In certain embodiments, the modification can include a thioether, e.g., beryl-S-tritylthiol (Manoharan et al., Ann. N. Y. Acad. Sci., 1992, 660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991, 10:1111-1118; Kabanov et al., FEBS Lett., 1990, 259:327-330; Svinarchuk et al., Biochimie, 1993, 75:49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids Res., 1990, 18:3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229-237), or an octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).

In certain embodiments, a ligand alters the distribution, targeting or lifetime of an iRNA agent into which it is incorporated. In preferred embodiments a ligand provides an enhanced affinity for a selected target, e.g., molecule, cell or cell type, compartment, e.g., a cellular or organ compartment, tissue, organ or region of the body, as, e.g., compared to a species absent such a ligand. Preferred ligands do not take part in duplex pairing in a duplexed nucleic acid.

Ligands can include a naturally occurring substance, such as a protein (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulin); carbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, N-acetylgalactosamine, or hyaluronic acid); or a lipid. The ligand can also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polyamino acid. Examples of polyamino acids include polyamino acid is a polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, or polyphosphazine. Example of polyamines include: polyethylenimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary salt of a polyamine, or an alpha helical peptide.

Ligands can also include targeting groups, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type such as a kidney cell. A targeting group can be a thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, Mucin carbohydrate, multivalent lactose, monovalent or multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucoseamine multivalent mannose, multivalent fucose, glycosylated polyaminoacids, transferrin, bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate, vitamin B12, vitamin A, biotin, or an RGD peptide or RGD peptide mimetic. In certain embodiments, ligands include monovalent or multivalent galactose. In certain embodiments, ligands include cholesterol.

Other examples of ligands include dyes, intercalating agents (e.g. acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA), lipophilic molecules, e.g., cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, bomeol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]₂, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin), transport/absorption facilitators (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.

Ligands can be proteins, e.g., glycoproteins, or peptides, e.g., molecules having a specific affinity for a co-ligand, or antibodies e.g., an antibody, that binds to a specified cell type such as a hepatic cell. Ligands can also include hormones and hormone receptors. They can also include non-peptidic species, such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose, or multivalent fucose. The ligand can be, for example, a lipopolysaccharide, an activator of p38 MAP kinase, or an activator of NF-κB.

The ligand can be a substance, e.g., a drug, which can increase the uptake of the iRNA agent into the cell, for example, by disrupting the cell's cytoskeleton, e.g., by disrupting the cell's microtubules, microfilaments, or intermediate filaments. The drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.

In some embodiments, a ligand attached to an iRNA as described herein acts as a pharmacokinetic modulator (PK modulator). PK modulators include lipophiles, bile acids, steroids, phospholipid analogues, peptides, protein binding agents, PEG, vitamins etc. Exemplary PK modulators include, but are not limited to, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkylglycerides, diacylglyceride, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin etc. Oligonucleotides that comprise a number of phosphorothioate linkages are also known to bind to serum protein, thus short oligonucleotides, e.g., oligonucleotides of about 5 bases, 10 bases, 15 bases, or 20 bases, comprising multiple of phosphorothioate linkages in the backbone are also amenable to the present invention as ligands (e.g. as PK modulating ligands). In addition, aptamers that bind serum components (e.g. serum proteins) are also suitable for use as PK modulating ligands in the embodiments described herein.

Ligand-conjugated iRNAs of the invention may be synthesized by the use of an oligonucleotide that bears a pendant reactive functionality, such as that derived from the attachment of a linking molecule onto the oligonucleotide (described below). This reactive oligonucleotide may be reacted directly with commercially-available ligands, ligands that are synthesized bearing any of a variety of protecting groups, or ligands that have a linking moiety attached thereto.

The oligonucleotides used in the conjugates of the present invention may be conveniently and routinely made through the well-known technique of solid-phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is also known to use similar techniques to prepare other oligonucleotides, such as the phosphorothioates and alkylated derivatives.

In the ligand-conjugated iRNAs and ligand-molecule bearing sequence-specific linked nucleosides of the present invention, the oligonucleotides and oligonucleosides may be assembled on a suitable DNA synthesizer utilizing standard nucleotide or nucleoside precursors, or nucleotide or nucleoside conjugate precursors that already bear the linking moiety, ligand-nucleotide or nucleoside-conjugate precursors that already bear the ligand molecule, or non-nucleoside ligand-bearing building blocks.

When using nucleotide-conjugate precursors that already bear a linking moiety, the synthesis of the sequence-specific linked nucleosides is typically completed, and the ligand molecule is then reacted with the linking moiety to form the ligand-conjugated oligonucleotide. In some embodiments, the oligonucleotides or linked nucleosides of the present invention are synthesized by an automated synthesizer using phosphoramidites derived from ligand-nucleoside conjugates in addition to the standard phosphoramidites and non-standard phosphoramidites that are commercially available and routinely used in oligonucleotide synthesis.

A. Lipid Conjugates

In certain embodiments, the ligand or conjugate is a lipid or lipid-based molecule. Such a lipid or lipid-based molecule preferably binds a serum protein, e.g., human serum albumin (HSA). An HSA binding ligand allows for distribution of the conjugate to a target tissue, e.g., a non-kidney target tissue of the body. For example, the target tissue can be the liver, including parenchymal cells of the liver. Other molecules that can bind HSA can also be used as ligands. For example, naproxen or aspirin can be used. A lipid or lipid-based ligand can (a) increase resistance to degradation of the conjugate, (b) increase targeting or transport into a target cell or cell membrane, or (c) can be used to adjust binding to a serum protein, e.g., HSA.

A lipid based ligand can be used to inhibit, e.g., control the binding of the conjugate to a target tissue. For example, a lipid or lipid-based ligand that binds to HSA more strongly will be less likely to be targeted to the kidney and therefore less likely to be cleared from the body. A lipid or lipid-based ligand that binds to HSA less strongly can be used to target the conjugate to the kidney.

In certain embodiments, the lipid based ligand binds HSA. Preferably, it binds HSA with a sufficient affinity such that the conjugate will be preferably distributed to a non-kidney tissue. However, it is preferred that the affinity not be so strong that the HSA-ligand binding cannot be reversed.

In other embodiments, the lipid based ligand binds HSA weakly or not at all, such that the conjugate will be preferably distributed to the kidney. Other moieties that target to kidney cells can also be used in place of, or in addition to, the lipid based ligand.

In another aspect, the ligand is a moiety, e.g., a vitamin, which is taken up by a target cell, e.g., a proliferating cell. These are particularly useful for treating disorders characterized by unwanted cell proliferation, e.g., of the malignant or non-malignant type, e.g., cancer cells. Exemplary vitamins include vitamin A, E, and K. Other exemplary vitamins include are B vitamin, e.g., folic acid, B12, riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up by target cells such as liver cells. Also included are HSA and low density lipoprotein (LDL).

B. Cell Permeation Agents

In another aspect, the ligand is a cell-permeation agent, preferably a helical cell-permeation agent. Preferably, the agent is amphipathic. An exemplary agent is a peptide such as tat or antennopedia. If the agent is a peptide, it can be modified, including a peptidylmimetic, invertomers, non-peptide or pseudo-peptide linkages, and use of D-amino acids. The helical agent is preferably an alpha-helical agent, which preferably has a lipophilic and a lipophobic phase.

The ligand can be a peptide or peptidomimetic. A peptidomimetic (also referred to herein as an oligopeptidomimetic) is a molecule capable of folding into a defined three-dimensional structure similar to a natural peptide. The attachment of peptide and peptidomimetics to iRNA agents can affect pharmacokinetic distribution of the iRNA, such as by enhancing cellular recognition and absorption. The peptide or peptidomimetic moiety can be about 5-50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.

A peptide or peptidomimetic can be, for example, a cell permeation peptide, cationic peptide, amphipathic peptide, or hydrophobic peptide (e.g., consisting primarily of Tyr, Trp, or Phe). The peptide moiety can be a dendrimer peptide, constrained peptide or crosslinked peptide. In another alternative, the peptide moiety can include a hydrophobic membrane translocation sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF having the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO:24). An RFGF analogue (e.g., amino acid sequence AALLPVLLAAP (SEQ ID NO:25) containing a hydrophobic MTS can also be a targeting moiety. The peptide moiety can be a “delivery” peptide, which can carry large polar molecules including peptides, oligonucleotides, and protein across cell membranes. For example, sequences from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO:26) and the Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO:27) have been found to be capable of functioning as delivery peptides. A peptide or peptidomimetic can be encoded by a random sequence of DNA, such as a peptide identified from a phage-display library, or one-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature, 354:82-84, 1991). Examples of a peptide or peptidomimetic tethered to a dsRNA agent via an incorporated monomer unit for cell targeting purposes is an arginine-glycine-aspartic acid (RGD)-peptide, or RGD mimic. A peptide moiety can range in length from about 5 amino acids to about 40 amino acids. The peptide moieties can have a structural modification, such as to increase stability or direct conformational properties. Any of the structural modifications described below can be utilized.

An RGD peptide for use in the compositions and methods of the invention may be linear or cyclic, and may be modified, e.g., glycosylated or methylated, to facilitate targeting to a specific tissue(s). RGD-containing peptides and peptidiomimemtics may include D-amino acids, as well as synthetic RGD mimics. In addition to RGD, one can use other moieties that target the integrin ligand. Preferred conjugates of this ligand target PECAM-1 or VEGF.

A “cell permeation peptide” is capable of permeating a cell, e.g., a microbial cell, such as a bacterial or fungal cell, or a mammalian cell, such as a human cell. A microbial cell-permeating peptide can be, for example, an α-helical linear peptide (e.g., LL-37 or Ceropin P1), a disulfide bond-containing peptide (e.g., α-defensin, β-defensin or bactenecin), or a peptide containing only one or two dominating amino acids (e.g., PR-39 or indolicidin). A cell permeation peptide can also include a nuclear localization signal (NLS). For example, a cell permeation peptide can be a bipartite amphipathic peptide, such as MPG, which is derived from the fusion peptide domain of HIV-1 gp41 and the NLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31:2717-2724, 2003).

C. Carbohydrate Conjugates

In some embodiments of the compositions and methods of the invention, an iRNA further comprises a carbohydrate. The carbohydrate conjugated iRNA is advantageous for the in vivo delivery of nucleic acids, as well as compositions suitable for in vivo therapeutic use, as described herein. As used herein, “carbohydrate” refers to a compound which is either a carbohydrate per se made up of one or more monosaccharide units having at least 6 carbon atoms (which can be linear, branched or cyclic) with an oxygen, nitrogen or sulfur atom bonded to each carbon atom; or a compound having as a part thereof a carbohydrate moiety made up of one or more monosaccharide units each having at least six carbon atoms (which can be linear, branched or cyclic), with an oxygen, nitrogen or sulfur atom bonded to each carbon atom. Representative carbohydrates include the sugars (mono-, di-, tri-, and oligosaccharides containing from about 4, 5, 6, 7, 8, or 9 monosaccharide units), and polysaccharides such as starches, glycogen, cellulose and polysaccharide gums. Specific monosaccharides include C5 and above (e.g., C5, C6, C7, or C8) sugars; di- and trisaccharides include sugars having two or three monosaccharide units (e.g., C5, C6, C7, or C8).

In certain embodiments, a carbohydrate conjugate for use in the compositions and methods of the invention is a monosaccharide.

In other embodiments, a carbohydrate conjugate for use in the compositions and methods of the invention is selected from the group:

In certain embodiments, the ligand is an N-acetylgalactosamine (GalNAc) or GalNAc derivative. In certain embodiments of the invention, the GalNAc or GalNAc derivative is attached to an iRNA agent of the invention via a monovalent linker. In some embodiments, the GalNAc or GalNAc derivative is attached to an iRNA agent of the invention via a bivalent linker. In yet other embodiments of the invention, the GalNAc or GalNAc derivative is attached to an iRNA agent of the invention via a trivalent linker.

In one embodiment, the double stranded RNAi agents of the invention comprise one GalNAc or GalNAc derivative attached to the iRNA agent. In another embodiment, the double stranded RNAi agents of the invention comprise a plurality (e.g., 2, 3, 4, 5, or 6) GalNAc or GalNAc derivatives, each independently attached to a plurality of nucleotides of the double stranded RNAi agent through a plurality of monovalent linkers.

In some embodiments, for example, when the two strands of an iRNA agent of the invention are part of one larger molecule connected by an uninterrupted chain of nucleotides between the 3′-end of one strand and the 5′-end of the respective other strand forming a hairpin loop comprising, a plurality of unpaired nucleotides, each unpaired nucleotide within the hairpin loop may independently comprise a GalNAc or GalNAc derivative attached via a monovalent linker. The hairpin loop may also be formed by an extended overhang in one strand of the duplex.

The GalNAc or GalNAc derivative may be conjugated to the 3′ end of the sense strand of the double stranded RNAi agent, the 5′ end of the sense strand of the double stranded RNAi agent, the 3′ end of the antisense strand of the double stranded RNAi agent, or the 5′ end of the antisense strand of the double stranded RNAi agent. In certain embodiments, the monosaccharide is an N-acetylgalactosamine, such as

Another representative carbohydrate conjugate for use in the embodiments described herein includes, but is not limited to,

(Formula XXIII), when one of X or Y is an oligonucleotide, the other is a hydrogen.

In some embodiments, the carbohydrate conjugate further comprises one or more additional ligands as described above, such as, but not limited to, a PK modulator or a cell permeation peptide.

Additional carbohydrate conjugates suitable for use in the present invention include those described in PCT Publication Nos. WO 2014/179620 and WO 2014/179627, the entire contents of each of which are incorporated herein by reference.

D. Linkers

In some embodiments, the conjugate or ligand described herein can be attached to an iRNA oligonucleotide with various linkers that can be cleavable or non-cleavable.

The term “linker” or “linking group” means an organic moiety that connects two parts of a compound, e.g., covalently attaches two parts of a compound. Linkers typically comprise a direct bond or an atom such as oxygen or sulfur, a unit such as NR8, C(O), C(O)NH, SO, SO₂, SO₂NH or a chain of atoms, such as, but not limited to, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylhererocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylhereroaryl, which one or more methylenes can be interrupted or terminated by O, S, S(O), SO₂, N(R8), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocyclic; where R8 is hydrogen, acyl, aliphatic, or substituted aliphatic. In one embodiment, the linker is between about 1-24 atoms, 2-24, 3-24, 4-24, 5-24, 6-24, 6-18, 7-18, 8-18, 7-17, 8-17, 6-16, 7-16, or 8-16 atoms.

A cleavable linking group is one which is sufficiently stable outside the cell, but which upon entry into a target cell is cleaved to release the two parts the linker is holding together. In a preferred embodiment, the cleavable linking group is cleaved at least about 10 times, 20, times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, or 100 times faster in a target cell or under a first reference condition (which can, e.g., be selected to mimic or represent intracellular conditions) than in the blood of a subject, or under a second reference condition (which can, e.g., be selected to mimic or represent conditions found in the blood or serum).

Cleavable linking groups are susceptible to cleavage agents, e.g., pH, redox potential, or the presence of degradative molecules. Generally, cleavage agents are more prevalent or found at higher levels or activities inside cells than in serum or blood. Examples of such degradative agents include: redox agents which are selected for particular substrates or which have no substrate specificity, including, e.g., oxidative or reductive enzymes or reductive agents such as mercaptans, present in cells, that can degrade a redox cleavable linking group by reduction; esterases; endosomes or agents that can create an acidic environment, e.g., those that result in a pH of five or lower; enzymes that can hydrolyze or degrade an acid cleavable linking group by acting as a general acid, peptidases (which can be substrate specific), and phosphatases.

A cleavable linkage group, such as a disulfide bond can be susceptible to pH. The pH of human serum is 7.4, while the average intracellular pH is slightly lower, ranging from about 7.1-7.3. Endosomes have a more acidic pH, in the range of 5.5-6.0, and lysosomes have an even more acidic pH at around 5.0. Some linkers will have a cleavable linking group that is cleaved at a preferred pH, thereby releasing a cationic lipid from the ligand inside the cell, or into the desired compartment of the cell.

A linker can include a cleavable linking group that is cleavable by a particular enzyme. The type of cleavable linking group incorporated into a linker can depend on the cell to be targeted. For example, a liver-targeting ligand can be linked to a cationic lipid through a linker that includes an ester group. Liver cells are rich in esterases, and therefore the linker will be cleaved more efficiently in liver cells than in cell types that are not esterase-rich. Other cell-types rich in esterases include cells of the lung, renal cortex, and testis.

Linkers that contain peptide bonds can be used when targeting cell types rich in peptidases, such as liver cells and synoviocytes.

In general, the suitability of a candidate cleavable linking group can be evaluated by testing the ability of a degradative agent (or condition) to cleave the candidate linking group. It will also be desirable to also test the candidate cleavable linking group for the ability to resist cleavage in the blood or when in contact with other non-target tissue. Thus, one can determine the relative susceptibility to cleavage between a first and a second condition, where the first is selected to be indicative of cleavage in a target cell and the second is selected to be indicative of cleavage in other tissues or biological fluids, e.g., blood or serum. The evaluations can be carried out in cell free systems, in cells, in cell culture, in organ or tissue culture, or in whole animals. It can be useful to make initial evaluations in cell-free or culture conditions and to confirm by further evaluations in whole animals. In preferred embodiments, useful candidate compounds are cleaved at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

i. Redox Cleavable Linking Groups

In certain embodiments, a cleavable linking group is a redox cleavable linking group that is cleaved upon reduction or oxidation. An example of reductively cleavable linking group is a disulphide linking group (—S—S—). To determine if a candidate cleavable linking group is a suitable “reductively cleavable linking group,” or for example is suitable for use with a particular iRNA moiety and particular targeting agent one can look to methods described herein. For example, a candidate can be evaluated by incubation with dithiothreitol (DTT), or other reducing agent using reagents know in the art, which mimic the rate of cleavage which would be observed in a cell, e.g., a target cell. The candidates can also be evaluated under conditions which are selected to mimic blood or serum conditions. In one, candidate compounds are cleaved by at most about 10% in the blood. In other embodiments, useful candidate compounds are degraded at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood (or under in vitro conditions selected to mimic extracellular conditions). The rate of cleavage of candidate compounds can be determined using standard enzyme kinetics assays under conditions chosen to mimic intracellular media and compared to conditions chosen to mimic extracellular media.

ii. Phosphate-Based Cleavable Linking Groups

In other embodiments, a cleavable linker comprises a phosphate-based cleavable linking group. A phosphate-based cleavable linking group is cleaved by agents that degrade or hydrolyze the phosphate group. An example of an agent that cleaves phosphate groups in cells are enzymes such as phosphatases in cells. Examples of phosphate-based linking groups are —O—P(O)(ORk)-O—, —O—P(S)(ORk)-O—, —O—P(S)(SRk)-O—, —S—P(O)(ORk)-O—, —O—P(O)(ORk)-S—, —S—P(O)(ORk)-S—, —O—P(S)(ORk)-S—, —S—P(S)(ORk)-O—, —O—P(O)(Rk)-O—, —O—P(S)(Rk)-O—, —S—P(O)(Rk)-O—, —S—P(S)(Rk)-O—, —S—P(O)(Rk)-S—, —O—P(S)(Rk)-S—. Preferred embodiments are —O—P(O)(OH)—O—, —O—P(S)(OH)—O—, —O—P(S)(SH)—O—, —S—P(O)(OH)—O—, —O—P(O)(OH)—S—, —S—P(O)(OH)—S—, —O—P(S)(OH)—S—, —S—P(S)(OH)—O—, —O—P(O)(H)—O—, —O—P(S)(H)—O—, —S—P(O)(H)—O, —S—P(S)(H)—O—, —S—P(O)(H)—S—, and —O—P(S)(H)—S—. A preferred embodiment is —O—P(O)(OH)—O—. These candidates can be evaluated using methods analogous to those described above.

iii. Acid Cleavable Linking Groups

In other embodiments, a cleavable linker comprises an acid cleavable linking group. An acid cleavable linking group is a linking group that is cleaved under acidic conditions. In preferred embodiments acid cleavable linking groups are cleaved in an acidic environment with a pH of about 6.5 or lower (e.g., about 6.0, 5.5, 5.0, or lower), or by agents such as enzymes that can act as a general acid. In a cell, specific low pH organelles, such as endosomes and lysosomes can provide a cleaving environment for acid cleavable linking groups. Examples of acid cleavable linking groups include but are not limited to hydrazones, esters, and esters of amino acids. Acid cleavable groups can have the general formula —C═NN—, C(O)O, or —OC(O). A preferred embodiment is when the carbon attached to the oxygen of the ester (the alkoxy group) is an aryl group, substituted alkyl group, or tertiary alkyl group such as dimethyl pentyl or t-butyl. These candidates can be evaluated using methods analogous to those described above.

iv. Ester-Based Linking Groups

In other embodiments, a cleavable linker comprises an ester-based cleavable linking group. An ester-based cleavable linking group is cleaved by enzymes such as esterases and amidases in cells. Examples of ester-based cleavable linking groups include, but are not limited to, esters of alkylene, alkenylene and alkynylene groups. Ester cleavable linking groups have the general formula —C(O)O—, or —OC(O)—. These candidates can be evaluated using methods analogous to those described above.

v. Peptide-Based Cleaving Groups

In yet other embodiments, a cleavable linker comprises a peptide-based cleavable linking group. A peptide-based cleavable linking group is cleaved by enzymes such as peptidases and proteases in cells. Peptide-based cleavable linking groups are peptide bonds formed between amino acids to yield oligopeptides (e.g., dipeptides, tripeptides etc.) and polypeptides. Peptide-based cleavable groups do not include the amide group (—C(O)NH—). The amide group can be formed between any alkylene, alkenylene or alkynelene. A peptide bond is a special type of amide bond formed between amino acids to yield peptides and proteins. The peptide based cleavage group is generally limited to the peptide bond (i.e., the amide bond) formed between amino acids yielding peptides and proteins and does not include the entire amide functional group. Peptide-based cleavable linking groups have the general formula—NHCHRAC(O)NHCHRBC(O)—, where RA and RB are the R groups of the two adjacent amino acids. These candidates can be evaluated using methods analogous to those described above.

In some embodiments, an iRNA of the invention is conjugated to a carbohydrate through a linker. Non-limiting examples of iRNA carbohydrate conjugates with linkers of the compositions and methods of the invention include, but are not limited to,

when one of X or Y is an oligonucleotide, the other is a hydrogen.

In certain embodiments of the compositions and methods of the invention, a ligand is one or more “GalNAc” (N-acetylgalactosamine) derivatives attached through a monovalent, a bivalent or a trivalent branched linker.

In certain embodiments, a dsRNA of the invention is conjugated to a monovalent, a bivalent or a trivalent branched linker selected from the group of structures shown in any of formula (XXXII)-(XXXV):

wherein:

q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B and q5C represent independently for each occurrence 0-20 and wherein the repeating unit can be the same or different;

P^(2A), P^(2B), P^(3A), P^(3B), P^(4A), P^(4B), P^(5A), P^(5B), P^(5C), T^(2A), T^(2B), T^(3A) T^(3B), T^(4A) T^(4B), T^(4A), T^(5B), T^(5C) are each independently for each occurrence absent, CO, NH, O, S, OC(O), NHC(O), CH₂, CH₂NH, or CH₂O;

Q^(2A), Q^(2B), Q^(3A), Q^(3B), Q^(4A), Q^(4B), Q^(5A), Q^(5B), Q^(5C) are independently for each occurrence absent, alkylene, substituted alkylene wherein one or more methylenes can be interrupted or terminated by one or more of O, S, S(O), SO₂, N(R^(N)), C(R′)═C(R″), C≡C, or C(O);

R^(2A), R^(2B), R^(3A), R^(3B), R^(4A), R^(4B), R^(5A), R^(5B), R^(5C) are each independently for each occurrence absent, NH, O, S, CH₂, C(O)O, C(O)NH, NHCH(R^(a))C(O), —C(O)—CH(R^(a))—NH—, CO, CH═N—O,

heterocyclyl;

L^(2A), L^(2B), L^(3A), L^(3B), L^(4A), L^(4B), L^(5A), L^(5B), and L^(5C) represent the ligand; i.e. each independently for each occurrence a monosaccharide (such as GalNAc), disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide; and R^(a) is H or amino acid side chain. Trivalent conjugating GalNAc derivatives are particularly useful for use with RNAi agents for inhibiting the expression of a target gene, such as those of formula (XXXV):

-   -   wherein L^(5A) L^(5B) and L^(5C) represent a monosaccharide,         such as GalNAc derivative.

Examples of suitable monovalent, bivalent and trivalent branched linker groups conjugating GalNAc derivatives include, but are not limited to, the structures recited above as formulas II, VII, XI, X, and XIII.

Representative US patents that teach the preparation of RNA conjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928; 5,688,941; 6,294,664; 6,320,017; 6,576,752; 6,783,931; 6,900,297; 7,037,646; and 8,106,022, the entire contents of each of which are hereby incorporated herein by reference.

It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the aforementioned modifications can be incorporated in a single compound or even at a single nucleoside within an iRNA. The present invention also includes iRNA compounds that are chimeric compounds.

“Chimeric” iRNA compounds or “chimeras,” in the context of this invention, are iRNA compounds, preferably dsRNAi agents, that contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of a dsRNA compound. These iRNAs typically contain at least one region wherein the RNA is modified so as to confer upon the iRNA increased resistance to nuclease degradation, increased cellular uptake, or increased binding affinity for the target nucleic acid. An additional region of the iRNA can serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of iRNA inhibition of gene expression. Consequently, comparable results can often be obtained with shorter iRNAs when chimeric dsRNAs are used, compared to phosphorothioate deoxy dsRNAs hybridizing to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.

In certain instances, the RNA of an iRNA can be modified by a non-ligand group. A number of non-ligand molecules have been conjugated to iRNAs in order to enhance the activity, cellular distribution or cellular uptake of the iRNA, and procedures for performing such conjugations are available in the scientific literature. Such non-ligand moieties have included lipid moieties, such as cholesterol (Kubo, T. et al., Biochem. Biophys. Res. Comm., 2007, 365(1):54-61; Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4:1053), athioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N. Y. Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3:2765), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993, 75:49), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990, 18:3777), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative United States patents that teach the preparation of such RNA conjugates have been listed above. Typical conjugation protocols involve the synthesis of RNAs bearing an aminolinker at one or more positions of the sequence. The amino group is then reacted with the molecule being conjugated using appropriate coupling or activating reagents. The conjugation reaction can be performed either with the RNA still bound to the solid support or following cleavage of the RNA, in solution phase. Purification of the RNA conjugate by HPLC typically affords the pure conjugate.

IV. Delivery of an iRNA of the Invention

The delivery of an iRNA of the invention to a cell e.g., a cell within a subject, such as a human subject (e.g., a subject in need thereof, such as a subject having a disease, disorder, or condition associated with IGFALS gene or IGF-1 gene expression) can be achieved in a number of different ways. For example, delivery may be performed by contacting a cell with an iRNA of the invention either in vitro or in vivo. In vivo delivery may also be performed directly by administering a composition comprising an iRNA, e.g., a dsRNA, to a subject. Alternatively, in vivo delivery may be performed indirectly by administering one or more vectors that encode and direct the expression of the iRNA. These alternatives are discussed further below.

In general, any method of delivering a nucleic acid molecule (in vitro or in vivo) can be adapted for use with an iRNA of the invention (see e.g., Akhtar S. and Julian R L. (1992) Trends Cell. Biol. 2(5):139-144 and WO94/02595, which are incorporated herein by reference in their entireties). For in vivo delivery, factors to consider in order to deliver an iRNA molecule include, for example, biological stability of the delivered molecule, prevention of non-specific effects, and accumulation of the delivered molecule in the target tissue. The non-specific effects of an iRNA can be minimized by local administration, for example, by direct injection or implantation into a tissue or topically administering the preparation. Local administration to a treatment site maximizes local concentration of the agent, limits the exposure of the agent to systemic tissues that can otherwise be harmed by the agent or that can degrade the agent, and permits a lower total dose of the iRNA molecule to be administered. Several studies have shown successful knockdown of gene products when a dsRNAi agent is administered locally. For example, intraocular delivery of a VEGF dsRNA by intravitreal injection in cynomolgus monkeys (Tolentino, M J, et al (2004) Retina 24:132-138) and subretinal injections in mice (Reich, S J., et al (2003)Mol. Vis. 9:210-216) were both shown to prevent neovascularization in an experimental model of age-related macular degeneration. In addition, direct intratumoral injection of a dsRNA in mice reduces tumor volume (Pille, J., et al (2005) Mol. Ther. 11:267-274) and can prolong survival of tumor-bearing mice (Kim, W J., et al (2006) Mol. Ther. 14:343-350; Li, S., et al (2007) Mol. Ther. 15:515-523). RNA interference has also shown success with local delivery to the CNS by direct injection (Dorn, G., et al. (2004) Nucleic Acids 32:e49; Tan, P H., et al (2005) Gene Ther. 12:59-66; Makimura, H., et al (2002) BMC Neurosci. 3:18; Shishkina, G T., et al (2004) Neuroscience 129:521-528; Thakker, E R., et al (2004) Proc. Natl. Acad. Sci. U.S.A. 101:17270-17275; Akaneya, Y., et al (2005) J Neurophysiol. 93:594-602) and to the lungs by intranasal administration (Howard, K A., et al (2006) Mol. Ther. 14:476-484; Zhang, X., et al (2004) J. Biol. Chem. 279:10677-10684; Bitko, V., et al (2005) Nat. Med. 11:50-55). For administering an iRNA systemically for the treatment of a disease, the RNA can be modified or alternatively delivered using a drug delivery system; both methods act to prevent the rapid degradation of the dsRNA by endo- and exo-nucleases in vivo. Modification of the RNA or the pharmaceutical carrier can also permit targeting of the iRNA to the target tissue and avoid undesirable off-target effects. iRNA molecules can be modified by chemical conjugation to lipophilic groups such as cholesterol to enhance cellular uptake and prevent degradation. For example, an iRNA directed against ApoB conjugated to a lipophilic cholesterol moiety was injected systemically into mice and resulted in knockdown of apoB mRNA in both the liver and jejunum (Soutschek, J., et al (2004) Nature 432:173-178). Conjugation of an iRNA to an aptamer has been shown to inhibit tumor growth and mediate tumor regression in a mouse model of prostate cancer (McNamara, J O, et al (2006) Nat. Biotechnol. 24:1005-1015). In an alternative embodiment, the iRNA can be delivered using drug delivery systems such as a nanoparticle, a dendrimer, a polymer, liposomes, or a cationic delivery system. Positively charged cationic delivery systems facilitate binding of an iRNA molecule (negatively charged) and also enhance interactions at the negatively charged cell membrane to permit efficient uptake of an iRNA by the cell. Cationic lipids, dendrimers, or polymers can either be bound to an iRNA, or induced to form a vesicle or micelle (see e.g., Kim S H, et al (2008) Journal of Controlled Release 129(2):107-116) that encases an iRNA. The formation of vesicles or micelles further prevents degradation of the iRNA when administered systemically. Methods for making and administering cationic-iRNA complexes are well within the abilities of one skilled in the art (see e.g., Sorensen, D R, et al (2003) J. Mol. Biol 327:761-766; Verma, U N, et al (2003) Clin. Cancer Res. 9:1291-1300; Arnold, A S et al (2007) J. Hypertens. 25:197-205, which are incorporated herein by reference in their entirety). Some non-limiting examples of drug delivery systems useful for systemic delivery of iRNAs include DOTAP (Sorensen, D R., et al (2003), supra; Verma, U N, et al (2003), supra), Oligofectamine, “solid nucleic acid lipid particles” (Zimmermann, T S, et al (2006) Nature 441:111-114), cardiolipin (Chien, P Y, et al (2005) Cancer Gene Ther. 12:321-328; Pal, A, et al (2005) Int J. Oncol. 26:1087-1091), polyethyleneimine (Bonnet M E, et al (2008) Pharm. Res. August 16 Epub ahead of print; Aigner, A. (2006) J. Biomed. Biotechnol. 71659), Arg-Gly-Asp (RGD) peptides (Liu, S. (2006) Mol. Pharm. 3:472-487), and polyamidoamines (Tomalia, D A, et al (2007) Biochem. Soc. Trans. 35:61-67; Yoo, H., et al (1999) Pharm. Res. 16:1799-1804). In some embodiments, an iRNA forms a complex with cyclodextrin for systemic administration. Methods for administration and pharmaceutical compositions of iRNAs and cyclodextrins can be found in U.S. Pat. No. 7,427,605, which is herein incorporated by reference in its entirety.

A. Vector Encoded iRNAs of the Invention

iRNA targeting an IGFALS gene or an IGF-1 gene can be expressed from transcription units inserted into DNA or RNA vectors (see, e.g., Couture, A, et al., TIG. (1996), 12:5-10; Skillern, A, et al., International PCT Publication No. WO 00/22113, Conrad, International PCT Publication No. WO 00/22114, and Conrad, U.S. Pat. No. 6,054,299). Expression can be transient (on the order of hours to weeks) or sustained (weeks to months or longer), depending upon the specific construct used and the target tissue or cell type. These transgenes can be introduced as a linear construct, a circular plasmid, or a viral vector, which can be an integrating or non-integrating vector. The transgene can also be constructed to permit it to be inherited as an extrachromosomal plasmid (Gassmann, et al., Proc. Natl. Acad. Sci. USA (1995) 92:1292).

The individual strand or strands of an iRNA can be transcribed from a promoter on an expression vector. Where two separate strands are to be expressed to generate, for example, a dsRNA, two separate expression vectors can be co-introduced (e.g., by transfection or infection) into a target cell. Alternatively each individual strand of a dsRNA can be transcribed by promoters both of which are located on the same expression plasmid. In one embodiment, a dsRNA is expressed as inverted repeat polynucleotides joined by a linker polynucleotide sequence such that the dsRNA has a stem and loop structure.

iRNA expression vectors are generally DNA plasmids or viral vectors. Expression vectors compatible with eukaryotic cells, preferably those compatible with vertebrate cells, can be used to produce recombinant constructs for the expression of an iRNA as described herein. Eukaryotic cell expression vectors are well known in the art and are available from a number of commercial sources. Typically, such vectors are provided containing convenient restriction sites for insertion of the desired nucleic acid segment. Delivery of iRNA expressing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from the patient followed by reintroduction into the patient, or by any other means that allows for introduction into a desired target cell.

Viral vector systems which can be utilized with the methods and compositions described herein include, but are not limited to, (a) adenovirus vectors; (b) retrovirus vectors, including but not limited to lentiviral vectors, moloney murine leukemia virus, etc.; (c) adeno-associated virus vectors; (d) herpes simplex virus vectors; (e) SV 40 vectors; (f) polyoma virus vectors; (g) papilloma virus vectors; (h) picornavirus vectors; (i) pox virus vectors such as an orthopox, e.g., vaccinia virus vectors or avipox, e.g. canary pox or fowl pox; and (j) a helper-dependent or gutless adenovirus. Replication-defective viruses can also be advantageous. Different vectors will or will not become incorporated into the cells' genome. The constructs can include viral sequences for transfection, if desired. Alternatively, the construct can be incorporated into vectors capable of episomal replication, e.g. EPV and EBV vectors. Constructs for the recombinant expression of an iRNA will generally require regulatory elements, e.g., promoters, enhancers, etc., to ensure the expression of the iRNA in target cells. Other aspects to consider for vectors and constructs are known in the art.

V. Pharmaceutical Compositions of the Invention

The present invention also includes pharmaceutical compositions and formulations which include the iRNAs of the invention. In one embodiment, provided herein are pharmaceutical compositions containing an iRNA, as described herein, and a pharmaceutically acceptable carrier. The pharmaceutical compositions containing the iRNA are useful for treating a disease or disorder associated with the expression or activity of an IGFALS gene or an IGF-1 gene. Such pharmaceutical compositions are formulated based on the mode of delivery. One example is compositions that are formulated for systemic administration via parenteral delivery, e.g., by subcutaneous (SC), intramuscular (IM), or intravenous (IV) delivery.

The pharmaceutical compositions of the invention may be administered in dosages sufficient to inhibit expression of an IGFALS gene or an IGF-1 gene. In general, a suitable dose of an iRNA of the invention will be in the range of about 0.001 to about 200.0 milligrams per kilogram body weight of the recipient per day, generally in the range of about 1 to 50 mg per kilogram body weight per day. Typically, a suitable dose of an iRNA of the invention will be in the range of about 0.1 mg/kg to about 5.0 mg/kg, preferably about 0.3 mg/kg and about 3.0 mg/kg. A repeat-dose regimine may include administration of a therapeutic amount of iRNA on a regular basis, such as every other day or once a year. In certain embodiments, the iRNA is administered about once per month to about once per quarter (i.e., about once every three months).

After an initial treatment regimen, the treatments can be administered on a less frequent basis. For example, after administration weekly or biweekly for three months, administration can be repeated once per month, for six months, or a year; or longer.

The pharmaceutical composition can be administered once daily, or the iRNA can be administered as two, three, or more sub-doses at appropriate intervals throughout the day or even using continuous infusion or delivery through a controlled release formulation. In that case, the iRNA contained in each sub-dose must be correspondingly smaller in order to achieve the total daily dosage. The dosage unit can also be compounded for delivery over several days, e.g., using a conventional sustained release formulation which provides sustained release of the iRNA over a several day period. Sustained release formulations are well known in the art and are particularly useful for delivery of agents at a particular site, such as could be used with the agents of the present invention. In this embodiment, the dosage unit contains a corresponding multiple of the daily dose.

In other embodiments, a single dose of the pharmaceutical compositions can be long lasting, such that subsequent doses are administered at not more than 3, 4, or 5 day intervals, or at not more than 1, 2, 3, or 4 week intervals. In some embodiments of the invention, a single dose of the pharmaceutical compositions of the invention is administered once per week. In other embodiments of the invention, a single dose of the pharmaceutical compositions of the invention is administered bi-monthly.

The skilled artisan will appreciate that certain factors can influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a composition can include a single treatment or a series of treatments. Estimates of effective dosages and in vivo half-lives for the individual iRNAs encompassed by the invention can be made using conventional methodologies or on the basis of in vivo testing using an appropriate animal model, as known in the art. For example, a mouse model of acromegaly was developed by Kovacs et al. (1997, Endocrinology) the entire contents of which are incorporated herein by reference. Bovine growth hormone transgenic mice also exhibit features of acromegaly (Palmiter et al., Science (1983), Olsson et al., Am J Phys Endo Metab (2003), Berryman et al, GH and IGF Res (2004), Izzard et al., GH and IGF Res (2009), Blutke et al., Mol and Cell Endo (2014)). Multiple animal models of cancer are known in the art.

The pharmaceutical compositions of the present invention can be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration can be topical (e.g., by a transdermal patch), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal, oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; subdermal, e.g., via an implanted device; or intracranial, e.g., by intraparenchymal, intrathecal or intraventricular administration.

The iRNA can be delivered in a manner to target a particular tissue (e.g., liver cells).

Pharmaceutical compositions and formulations for topical or transdermal administration can include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like can be necessary or desirable. Coated condoms, gloves and the like can also be useful. Suitable topical formulations include those in which the iRNAs featured in the invention are in admixture with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants. Suitable lipids and liposomes include neutral (e.g., dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline) negative (e.g., dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g., dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidyl ethanolamine DOTMA). iRNAs featured in the invention can be encapsulated within liposomes or can form complexes thereto, in particular to cationic liposomes. Alternatively, iRNAs can be complexed to lipids, in particular to cationic lipids. Suitable fatty acids and esters include but are not limited to arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a C₁₋₂₀ alkyl ester (e.g., isopropylmyristate IPM), monoglyceride, diglyceride or pharmaceutically acceptable salt thereof). Topical formulations are described in detail in U.S. Pat. No. 6,747,014, which is incorporated herein by reference.

A. iRNA Formulations Comprising Membranous Molecular Assemblies

An iRNA for use in the compositions and methods of the invention can be formulated for delivery in a membranous molecular assembly, e.g., a liposome or a micelle. As used herein, the term “liposome” refers to a vesicle composed of amphiphilic lipids arranged in at least one bilayer, e.g., one bilayer or a plurality of bilayers. Liposomes include unilamellar and multilamellar vesicles that have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the iRNA. The lipophilic material isolates the aqueous interior from an aqueous exterior, which typically does not include the iRNA composition, although in some examples, it may. Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Because the liposomal membrane is structurally similar to biological membranes, when liposomes are applied to a tissue, the liposomal bilayer fuses with bilayer of the cellular membranes. As the merging of the liposome and cell progresses, the internal aqueous contents that include the iRNA are delivered into the cell where the iRNA can specifically bind to a target RNA and can mediate RNA interference. In some cases the liposomes are also specifically targeted, e.g., to direct the iRNA to particular cell types.

A liposome containing an iRNA agent can be prepared by a variety of methods. In one example, the lipid component of a liposome is dissolved in a detergent so that micelles are formed with the lipid component. For example, the lipid component can be an amphipathic cationic lipid or lipid conjugate. The detergent can have a high critical micelle concentration and may be nonionic. Exemplary detergents include cholate, CHAPS, octylglucoside, deoxycholate, and lauroyl sarcosine. The iRNA agent preparation is then added to the micelles that include the lipid component. The cationic groups on the lipid interact with the iRNA agent and condense around the iRNA agent to form a liposome. After condensation, the detergent is removed, e.g., by dialysis, to yield a liposomal preparation of iRNA agent.

If necessary a carrier compound that assists in condensation can be added during the condensation reaction, e.g., by controlled addition. For example, the carrier compound can be a polymer other than a nucleic acid (e.g., spermine or spermidine). pH can also adjusted to favor condensation.

Methods for producing stable polynucleotide delivery vehicles, which incorporate a polynucleotide/cationic lipid complex as structural components of the delivery vehicle, are further described in, e.g., WO 96/37194, the entire contents of which are incorporated herein by reference. Liposome formation can also include one or more aspects of exemplary methods described in Felgner, P. L. et al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987; U.S. Pat. Nos. 4,897,355; 5,171,678; Bangham, et al. M Mol. Biol. 23:238, 1965; Olson, et al. Biochim. Biophys. Acta 557:9, 1979; Szoka, et al. Proc. Natl. Acad. Sci. 75: 4194, 1978; Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984; Kim, et al. Biochim. Biophys. Acta 728:339, 1983; and Fukunaga, et al. Endocrinol. 115:757, 1984. Commonly used techniques for preparing lipid aggregates of appropriate size for use as delivery vehicles include sonication and freeze-thaw plus extrusion (see, e.g., Mayer, et al. Biochim. Biophys. Acta 858:161, 1986). Microfluidization can be used when consistently small (50 to 200 nm) and relatively uniform aggregates are desired (Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984). These methods are readily adapted to packaging iRNA agent preparations into liposomes.

Liposomes fall into two broad classes. Cationic liposomes are positively charged liposomes which interact with the negatively charged nucleic acid molecules to form a stable complex. The positively charged nucleic acid/liposome complex binds to the negatively charged cell surface and is internalized in an endosome. Due to the acidic pH within the endosome, the liposomes are ruptured, releasing their contents into the cell cytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).

Liposomes which are pH-sensitive or negatively-charged, entrap nucleic acids rather than complex with it. Since both the nucleic acid and the lipid are similarly charged, repulsion rather than complex formation occurs. Nevertheless, some nucleic acid is entrapped within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver nucleic acids encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was detected in the target cells (Zhou et al., Journal of Controlled Release, 1992, 19, 269-274).

One major type of liposomal composition includes phospholipids other than naturally-derived phosphatidylcholine. Neutral liposome compositions, for example, can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions generally are formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are formed primarily from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposomal composition is formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC. Another type is formed from mixtures of two or more of phospholipid, phosphatidylcholine, and cholesterol.

Examples of other methods to introduce liposomes into cells in vitro and in vivo include U.S. Pat. Nos. 5,283,185 and 5,171,678; WO 94/00569; WO 93/24640; WO 91/16024; Felgner, J. Biol. Chem. 269:2550, 1994; Nabel, Proc. Natl. Acad. Sci. 90:11307, 1993; Nabel, Human Gene Ther. 3:649, 1992; Gershon, Biochem. 32:7143, 1993; and Strauss EMBO J 11:417, 1992.

Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol. Non-ionic liposomal formulations comprising Novasome™ I (glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver cyclosporin-A into the dermis of mouse skin. Results indicated that such non-ionic liposomal systems were effective in facilitating the deposition of cyclosporine A into different layers of the skin (Hu et al. S. T. P. Pharma. Sci., 1994, 4(6) 466).

Liposomes also include “sterically stabilized” liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome (A) comprises one or more glycolipids, such as monosialoganglioside G_(M1), or (B) is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. While not wishing to be bound by any particular theory, it is thought in the art that, at least for sterically stabilized liposomes containing gangliosides, sphingomyelin, or PEG-derivatized lipids, the enhanced circulation half-life of these sterically stabilized liposomes derives from a reduced uptake into cells of the reticuloendothelial system (RES) (Allen et al., FEBS Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

Various liposomes comprising one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64) reported the ability of monosialoganglioside G_(M1), galactocerebroside sulfate and phosphatidylinositol to improve blood half-lives of liposomes. These findings were expounded upon by Gabizon et al. (Proc. Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO 88/04924, both to Allen et al., disclose liposomes comprising (1) sphingomyelin and (2) the ganglioside G_(M1) or a galactocerebroside sulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomes comprising sphingomyelin. Liposomes comprising 1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Lim et al).

In some embodiments, cationic liposomes are used. Cationic liposomes possess the advantage of being able to fuse to the cell membrane. Non-cationic liposomes, although not able to fuse as efficiently with the plasma membrane, are taken up by macrophages in vivo and can be used to deliver iRNA agents to macrophages.

Further advantages of liposomes include: liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water and lipid soluble drugs; liposomes can protect encapsulated iRNAs in their internal compartments from metabolism and degradation (Rosoff, in “Pharmaceutical Dosage Forms,” Lieberman, Rieger and Banker (Eds.), 1988, volume 1, p. 245). Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size, and the aqueous volume of the liposomes.

A positively charged synthetic cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) can be used to form small liposomes that interact spontaneously with nucleic acid to form lipid-nucleic acid complexes which are capable of fusing with the negatively charged lipids of the cell membranes of tissue culture cells, resulting in delivery of iRNA agent (see, e.g., Felgner, P. L. et al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987 and U.S. Pat. No. 4,897,355 for a description of DOTMA and its use with DNA).

A DOTMA analogue, 1,2-bis(oleoyloxy)-3-(trimethylammonia)propane (DOTAP) can be used in combination with a phospholipid to form DNA-complexing vesicles. Lipofectin™ (Bethesda Research Laboratories, Gaithersburg, Md.) is an effective agent for the delivery of highly anionic nucleic acids into living tissue culture cells that comprise positively charged DOTMA liposomes which interact spontaneously with negatively charged polynucleotides to form complexes. When enough positively charged liposomes are used, the net charge on the resulting complexes is also positive. Positively charged complexes prepared in this way spontaneously attach to negatively charged cell surfaces, fuse with the plasma membrane, and efficiently deliver functional nucleic acids into, for example, tissue culture cells. Another commercially available cationic lipid, 1,2-bis(oleoyloxy)-3,3-(trimethylammonia)propane (“DOTAP”) (Boehringer Mannheim, Indianapolis, Ind.) differs from DOTMA in that the oleoyl moieties are linked by ester, rather than ether linkages.

Other reported cationic lipid compounds include those that have been conjugated to a variety of moieties including, for example, carboxyspermine which has been conjugated to one of two types of lipids and includes compounds such as 5-carboxyspermylglycine dioctaoleoylamide (“DOGS”) (Transfectam™, Promega, Madison, Wis.) and dipalmitoylphosphatidylethanolamine 5-carboxyspermyl-amide (“DPPES”) (see, e.g., U.S. Pat. No. 5,171,678).

Another cationic lipid conjugate includes derivatization of the lipid with cholesterol (“DC-Chol”) which has been formulated into liposomes in combination with DOPE (See, Gao, X. and Huang, L., Biochim. Biophys. Res. Commun. 179:280, 1991). Lipopolylysine, made by conjugating polylysine to DOPE, has been reported to be effective for transfection in the presence of serum (Zhou, X. et al., Biochim. Biophys. Acta 1065:8, 1991). For certain cell lines, these liposomes containing conjugated cationic lipids, are said to exhibit lower toxicity and provide more efficient transfection than the DOTMA-containing compositions. Other commercially available cationic lipid products include DMRIE and DMRIE-HP (Vical, La Jolla, Calif.) and Lipofectamine (DOSPA) (Life Technology, Inc., Gaithersburg, Md.). Other cationic lipids suitable for the delivery of oligonucleotides are described in WO 98/39359 and WO 96/37194.

Liposomal formulations are particularly suited for topical administration, liposomes present several advantages over other formulations. Such advantages include reduced side effects related to high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer iRNA agent into the skin. In some implementations, liposomes are used for delivering iRNA agent to epidermal cells and also to enhance the penetration of iRNA agent into dermal tissues, e.g., into skin. For example, the liposomes can be applied topically. Topical delivery of drugs formulated as liposomes to the skin has been documented (see, e.g., Weiner et al., Journal of Drug Targeting, 1992, vol. 2, 405-410 and du Plessis et al., Antiviral Research, 18, 1992, 259-265; Mannino, R. J. and Fould-Fogerite, S., Biotechniques 6:682-690, 1988; Itani, T. et al. Gene 56:267-276. 1987; Nicolau, C. et al. Meth. Enz. 149:157-176, 1987; Straubinger, R. M. and Papahadjopoulos, D. Meth. Enz. 101:512-527, 1983; Wang, C. Y. and Huang, L., Proc. Natl. Acad. Sci. USA 84:7851-7855, 1987).

Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol. Non-ionic liposomal formulations comprising Novasome™ I (glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver a drug into the dermis of mouse skin. Such formulations with iRNA agent are useful for treating a dermatological disorder.

Liposomes that include iRNA can be made highly deformable. Such deformability can enable the liposomes to penetrate through pore that are smaller than the average radius of the liposome. For example, transfersomes are a type of deformable liposomes. Transferosomes can be made by adding surface edge activators, usually surfactants, to a standard liposomal composition. Transfersomes that include iRNAs can be delivered, for example, subcutaneously by infection in order to deliver iRNAs to keratinocytes in the skin. In order to cross intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter less than 50 nm, under the influence of a suitable transdermal gradient. In addition, due to the lipid properties, these transferosomes can be self-optimizing (adaptive to the shape of pores, e.g., in the skin), self-repairing, and can frequently reach their targets without fragmenting, and often self-loading.

Other formulations amenable to the present invention are described in WO/2008/042973.

Transfersomes are yet another type of liposomes, and are highly deformable lipid aggregates which are attractive candidates for drug delivery vehicles. Transfersomes can be described as lipid droplets which are so highly deformable that they are easily able to penetrate through pores which are smaller than the droplet. Transfersomes are adaptable to the environment in which they are used, e.g., they are self-optimizing (adaptive to the shape of pores in the skin), self-repairing, frequently reach their targets without fragmenting, and often self-loading. To make transfersomes it is possible to add surface edge-activators, usually surfactants, to a standard liposomal composition. Transfersomes have been used to deliver serum albumin to the skin. The transfersome-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a solution containing serum albumin.

Surfactants find wide application in formulations such as emulsions (including microemulsions) and liposomes. The most common way of classifying and ranking the properties of the many different types of surfactants, both natural and synthetic, is by the use of the hydrophile/lipophile balance (HLB). The nature of the hydrophilic group (also known as the “head”) provides the most useful means for categorizing the different surfactants used in formulations (Rieger, in “Pharmaceutical Dosage Forms”, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

If the surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants find wide application in pharmaceutical and cosmetic products and are usable over a wide range of pH values. In general their HLB values range from 2 to about 18 depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylated block polymers are also included in this class. The polyoxyethylene surfactants are the most popular members of the nonionic surfactant class.

If the surfactant molecule carries a negative charge when it is dissolved or dispersed in water, the surfactant is classified as anionic. Anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates. The most important members of the anionic surfactant class are the alkyl sulfates and the soaps.

If the surfactant molecule carries a positive charge when it is dissolved or dispersed in water, the surfactant is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. The quaternary ammonium salts are the most used members of this class.

If the surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines and phosphatides.

The use of surfactants in drug products, formulations and in emulsions has been reviewed (Rieger, in “Pharmaceutical Dosage Forms”, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

The iRNA for use in the methods of the invention can also be provided as micellar formulations. “Micelles” are defined herein as a particular type of molecular assembly in which amphipathic molecules are arranged in a spherical structure such that all the hydrophobic portions of the molecules are directed inward, leaving the hydrophilic portions in contact with the surrounding aqueous phase. The converse arrangement exists if the environment is hydrophobic.

A mixed micellar formulation suitable for delivery through transdermal membranes may be prepared by mixing an aqueous solution of iRNA, an alkali metal C₈ to C₂₂ alkyl sulphate, and a micelle forming compounds. Exemplary micelle forming compounds include lecithin, hyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid, glycolic acid, lactic acid, chamomile extract, cucumber extract, oleic acid, linoleic acid, linolenic acid, monoolein, monooleates, monolaurates, borage oil, evening of primrose oil, menthol, trihydroxy oxo cholanyl glycine and pharmaceutically acceptable salts thereof, glycerin, polyglycerin, lysine, polylysine, triolein, polyoxyethylene ethers and analogues thereof, polidocanol alkyl ethers and analogues thereof, chenodeoxycholate, deoxycholate, and mixtures thereof. The micelle forming compounds may be added at the same time or after addition of the alkali metal alkyl sulphate. Mixed micelles will form with substantially any kind of mixing of the ingredients but vigorous mixing in order to provide smaller size micelles.

In one method a first micellar composition is prepared which contains the RNAi and at least the alkali metal alkyl sulphate. The first micellar composition is then mixed with at least three micelle forming compounds to form a mixed micellar composition. In another method, the micellar composition is prepared by mixing the RNAi, the alkali metal alkyl sulphate and at least one of the micelle forming compounds, followed by addition of the remaining micelle forming compounds, with vigorous mixing.

Phenol or m-cresol may be added to the mixed micellar composition to stabilize the formulation and protect against bacterial growth. Alternatively, phenol or m-cresol may be added with the micelle forming ingredients. An isotonic agent such as glycerin may also be added after formation of the mixed micellar composition.

For delivery of the micellar formulation as a spray, the formulation can be put into an aerosol dispenser and the dispenser is charged with a propellant. The propellant, which is under pressure, is in liquid form in the dispenser. The ratios of the ingredients are adjusted so that the aqueous and propellant phases become one, i.e., there is one phase. If there are two phases, it is necessary to shake the dispenser prior to dispensing a portion of the contents, e.g., through a metered valve. The dispensed dose of pharmaceutical agent is propelled from the metered valve in a fine spray.

Propellants may include hydrogen-containing chlorofluorocarbons, hydrogen-containing fluorocarbons, dimethyl ether and diethyl ether. In certain embodiments, HFA 134a (1,1,1,2 tetrafluoroethane) may be used.

The specific concentrations of the essential ingredients can be determined by relatively straightforward experimentation. For absorption through the oral cavities, it is often desirable to increase, e.g., at least double or triple, the dosage for through injection or administration through the gastrointestinal tract.

B. Lipid Particles

iRNAs, e.g., dsRNAi agents of the invention may be fully encapsulated in a lipid formulation, e.g., a LNP, or other nucleic acid-lipid particle.

As used herein, the term “LNP” refers to a stable nucleic acid-lipid particle. LNPs typically contain a cationic lipid, a non-cationic lipid, and a lipid that prevents aggregation of the particle (e.g., a PEG-lipid conjugate). LNPs are extremely useful for systemic applications, as they exhibit extended circulation lifetimes following intravenous (i.v.) injection and accumulate at distal sites (e.g., sites physically separated from the administration site). LNPs include “pSPLP,” which include an encapsulated condensing agent-nucleic acid complex as set forth in PCT Publication No. WO 00/03683. The particles of the present invention typically have a mean diameter of about 50 nm to about 150 nm, more typically about 60 nm to about 130 nm, more typically about 70 nm to about 110 nm, most typically about 70 nm to about 90 nm, and are substantially nontoxic. In addition, the nucleic acids when present in the nucleic acid-lipid particles of the present invention are resistant in aqueous solution to degradation with a nuclease. Nucleic acid-lipid particles and their method of preparation are disclosed in, e.g., U.S. Pat. Nos. 5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432; US Publication No. 2010/0324120 and PCT Publication No. WO 96/40964.

In one embodiment, the lipid to drug ratio (mass/mass ratio) (e.g., lipid to dsRNA ratio) will be in the range of from about 1:1 to about 50:1, from about 1:1 to about 25:1, from about 3:1 to about 15:1, from about 4:1 to about 10:1, from about 5:1 to about 9:1, or about 6:1 to about 9:1. Ranges intermediate to the above recited ranges are also contemplated to be part of the invention.

The cationic lipid can be, for example, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N—(I-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N—(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), 1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-Dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA), 1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or 3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-Dioleylamino)-1,2-propanedio (DOAP), 1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) or analogs thereof, (3aR,5s,6aS)—N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine (ALN100), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (MC3), 1,1′-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol (Tech G1), or a mixture thereof. The cationic lipid can comprise from about 20 mol % to about 50 mol % or about 40 mol % of the total lipid present in the particle.

In some embodiments, the compound 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane can be used to prepare lipid-siRNA nanoparticles.

In some embodiments, the lipid-siRNA particle includes 40% 2, 2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane: 10% DSPC: 40% Cholesterol: 10% PEG-C-DOMG (mole percent) with a particle size of 63.0±20 nm and a 0.027 siRNA/Lipid Ratio.

The ionizable/non-cationic lipid can be an anionic lipid or a neutral lipid including, but not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, or a mixture thereof. The non-cationic lipid can be from about 5 mol % to about 90 mol %, about 10 mol %, or about 58 mol % if cholesterol is included, of the total lipid present in the particle.

The conjugated lipid that inhibits aggregation of particles can be, for example, a polyethyleneglycol (PEG)-lipid including, without limitation, a PEG-diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), a PEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof. The PEG-DAA conjugate can be, for example, a PEG-dilauryloxypropyl (Ci₂), a PEG-dimyristyloxypropyl (Ci₄), a PEG-dipalmityloxypropyl (Ci₆), or a PEG-distearyloxypropyl (C]₈). The conjugated lipid that prevents aggregation of particles can be from 0 mol % to about 20 mol % or about 2 mol % of the total lipid present in the particle.

In some embodiments, the nucleic acid-lipid particle further includes cholesterol at, e.g., about 10 mol % to about 60 mol % or about 48 mol % of the total lipid present in the particle.

In one embodiment, the lipidoid ND98-4HCl (MW 1487) (see US20090023673, which is incorporated herein by reference), Cholesterol (Sigma-Aldrich), and PEG-Ceramide C16 (Avanti Polar Lipids) can be used to prepare lipid-dsRNA nanoparticles (i.e., LNP01 particles). Stock solutions of each in ethanol can be prepared as follows: ND98, 133 mg/ml; Cholesterol, 25 mg/ml, PEG-Ceramide C16, 100 mg/ml. The ND98, Cholesterol, and PEG-Ceramide C16 stock solutions can then be combined in a, e.g., 42:48:10 molar ratio. The combined lipid solution can be mixed with aqueous dsRNA (e.g., in sodium acetate pH 5) such that the final ethanol concentration is about 35-45% and the final sodium acetate concentration is about 100-300 mM. Lipid-dsRNA nanoparticles typically form spontaneously upon mixing. Depending on the desired particle size distribution, the resultant nanoparticle mixture can be extruded through a polycarbonate membrane (e.g., 100 nm cut-off) using, for example, a thermobarrel extruder, such as Lipex Extruder (Northern Lipids, Inc). In some cases, the extrusion step can be omitted. Ethanol removal and simultaneous buffer exchange can be accomplished by, for example, dialysis or tangential flow filtration. Buffer can be exchanged with, for example, phosphate buffered saline (PBS) at about pH 7, e.g., about pH 6.9, about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, or about pH 7.4.

LNP01 formulations are described, e.g., in International Application Publication No. WO 2008/042973, which is hereby incorporated by reference.

Additional exemplary lipid-dsRNA formulations are described in Table 1.

TABLE 1 Exemplary lipid formulations cationic lipid/non-cationic lipid/cholesterol/PEG-lipid conjugate Ionizable/Cationic Lipid Lipid:siRNA ratio SNALP-1 1,2-Dilinolenyloxy-N,N- DLinDMA/DPPC/Cholesterol/PEG-cDMA dimethylaminopropane (DLinDMA) (57.1/7.1/34.4/1.4) lipid:siRNA~7:1 2-XTC 2,2-Dilinoleyl-4-dimethylaminoethyl- XTC/DPPC/Cholesterol/PEG-cDMA [1,3]-dioxolane (XTC) 57.1/7.1/34.4/1.4 lipid:siRNA 7:1 LNP05 2,2-Dilinoleyl-4-dimethylaminoethyl- XTC/DSPC/Cholesterol/PEG-DMG [1,3]-dioxolane (XTC) 57.5/7.5/31.5/3.5 lipid:siRNA~6:1 LNP06 2,2-Dilinoleyl-4-dimethylaminoethyl- XTC/DSPC/Cholesterol/PEG-DMG [1,3]-dioxolane (XTC) 57.5/7.5/31.5/3.5 lipid:siRNA~11:1 LNP07 2,2-Dilinoleyl-4-dimethylaminoethyl- XTC/DSPC/Cholesterol/PEG-DMG [1,3]-dioxolane (XTC) 60/7.5/31/1.5, lipid:siRNA 6:1 LNP08 2,2-Dilinoleyl-4-dimethylaminoethyl- XTC/DSPC/Cholesterol/PEG-DMG [1,3]-dioxolane (XTC) 60/7.5/31/1.5, lipid:siRNA~11:1 LNP09 2,2-Dilinoleyl-4-dimethylaminoethyl- XTC/DSPC/Cholesterol/PEG-DMG [1,3]-dioxolane (XTC) 50/10/38.5/1.5 Lipid:siRNA 10:1 LNP10 (3aR,5s,6aS)-N,N-dimethyl-2,2- ALN100/DSPC/Cholesterol/PEG-DMG di((9Z,12Z)-octadeca-9,12- 50/10/38.5/1.5 dienyl)tetrahydro-3aH- Lipid:siRNA 10:1 cyclopenta[d][1,3]dioxol-5-amine (ALN100) LNP11 (6Z,9Z,28Z,31Z)-heptatriaconta- MC-3/DSPC/Cholesterol/PEG-DMG 6,9,28,31-tetraen-19-yl 4- 50/10/38.5/1.5 (dimethylamino)butanoate (MC3) Lipid:siRNA 10:1 LNP12 1,1′-(2-(4-(2-((2-(bis(2- Tech G1/DSPC/Cholesterol/PEG-DMG hydroxydodecyl)amino)ethyl)(2- 50/10/38.5/1.5 hydroxydodecyl)amino)ethyl)piperazin- Lipid:siRNA 10:1 1-yl)ethylazanediyl)didodecan-2-ol (Tech G1) LNP13 XTC XTC/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 33:1 LNP14 MC3 MC3/DSPC/Chol/PEG-DMG 40/15/40/5 Lipid:siRNA: 11:1 LNP15 MC3 MC3/DSPC/Chol/PEG-DSG/ GalNAc-PEG-DSG 50/10/35/4.5/0.5 Lipid:siRNA: 11:1 LNP16 MC3 MC3/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 7:1 LNP17 MC3 MC3/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:siRNA: 10:1 LNP18 MC3 MC3/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 12:1 LNP19 MC3 MC3/DSPC/Chol/PEG-DMG 50/10/35/5 Lipid:siRNA: 8:1 LNP20 MC3 MC3/DSPC/Chol/PEG-DPG 50/10/38.5/1.5 Lipid:siRNA: 10:1 LNP21 C12-200 C12-200/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:siRNA: 7:1 LNP22 XTC XTC/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:siRNA: 10:1 DSPC: distearoylphosphatidylcholine DPPC: dipalmitoylphosphatidylcholine PEG-DMG: PEG-didimyristoyl glycerol (C14-PEG, or PEG-C14) (PEG with avg mol wt of 2000) PEG-DSG: PEG-distyryl glycerol (C18-PEG, or PEG-C18) (PEG with avg mol wt of 2000) PEG-cDMA: PEG-carbamoyl-1,2-dimyristyloxypropylamine (PEG with avg mol wt of 2000) SNALP (1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA)) comprising formulations are described in International Publication No. WO2009/127060, filed Apr. 15, 2009, which is hereby incorporated by reference.

XTC comprising formulations are described, e.g., in International Application No. PCT/US2010/022614, filed Jan. 29, 2010, which is hereby incorporated by reference.

MC3 comprising formulations are described, e.g., in US Patent Publication No. 2010/0324120, filed Jun. 10, 2010, the entire contents of which are hereby incorporated by reference.

ALNY-100 comprising formulations are described, e.g., International patent application number PCT/US09/63933, filed on Nov. 10, 2009, which is hereby incorporated by reference.

C12-200 comprising formulations are described in WO2010/129709, which is hereby incorporated by reference.

Compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions, or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids, or binders can be desirable. In some embodiments, oral formulations are those in which dsRNAs featured in the invention are administered in conjunction with one or more penetration enhancer surfactants and chelators. Suitable surfactants include fatty acids or esters or salts thereof, bile acids or salts thereof. Suitable bile acids/salts include chenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate. Suitable fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcamitine, an acylcholine, or a monoglyceride, a diglyceride or a pharmaceutically acceptable salt thereof (e.g., sodium). In some embodiments, combinations of penetration enhancers are used, for example, fatty acids/salts in combination with bile acids/salts. One exemplary combination is the sodium salt of lauric acid, capric acid and UDCA. Further penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. DsRNAs featured in the invention can be delivered orally, in granular form including sprayed dried particles, or complexed to form micro or nanoparticles. DsRNA complexing agents include poly-amino acids; polyimines; polyacrylates; polyalkylacrylates, polyoxethanes, polyalkylcyanoacrylates; cationized gelatins, albumins, starches, acrylates, polyethyleneglycols (PEG), and starches; polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans, celluloses, and starches. Suitable complexing agents include chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine, polyspermines, protamine, polyvinylpyridine, polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g., p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate), poly(butylcyanoacrylate), poly(isobutylcyanoacrylate), poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate, DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate, polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolic acid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulations for dsRNAs and their preparation are described in detail in U.S. Pat. No. 6,887,906, US Publn. No. 20030027780, and U.S. Pat. No. 6,747,014, each of which is incorporated herein by reference.

Compositions and formulations for parenteral, intraparenchymal (into the brain), intrathecal, intraventricular, or intrahepatic administration can include sterile aqueous solutions which can also contain buffers, diluents, and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds, and other pharmaceutically acceptable carriers or excipients.

Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions can be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids, and self-emulsifying semisolids. Formulations include those that target the liver when treating hepatic disorders such as hepatic carcinoma.

The pharmaceutical formulations of the present invention, which can conveniently be presented in unit dosage form, can be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

The compositions of the present invention can be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention can also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions can further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol, or dextran. The suspension can also contain stabilizers.

C. Additional Formulations

i. Emulsions

The iRNAs of the present invention can be prepared and formulated as emulsions. Emulsions are typically heterogeneous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1p m in diameter (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335; Higuchi et al., in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 301). Emulsions are often biphasic systems comprising two immiscible liquid phases intimately mixed and dispersed with each other. In general, emulsions can be of either the water-in-oil (w/o) or the oil-in-water (o/w) variety. When an aqueous phase is finely divided into and dispersed as minute droplets into a bulk oily phase, the resulting composition is called a water-in-oil (w/o) emulsion. Alternatively, when an oily phase is finely divided into and dispersed as minute droplets into a bulk aqueous phase, the resulting composition is called an oil-in-water (o/w) emulsion. Emulsions can contain additional components in addition to the dispersed phases, and the active drug which can be present as a solution either in the aqueous phase, oily phase or itself as a separate phase. Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and antioxidants can also be present in emulsions as needed. Pharmaceutical emulsions can also be multiple emulsions that are comprised of more than two phases such as, for example, in the case of oil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions. Such complex formulations often provide certain advantages that simple binary emulsions do not. Multiple emulsions in which individual oil droplets of an o/w emulsion enclose small water droplets constitute a w/o/w emulsion. Likewise a system of oil droplets enclosed in globules of water stabilized in an oily continuous phase provides an o/w/o emulsion.

Emulsions are characterized by little or no thermodynamic stability. Often, the dispersed or discontinuous phase of the emulsion is well dispersed into the external or continuous phase and maintained in this form through the means of emulsifiers or the viscosity of the formulation. Either of the phases of the emulsion can be a semisolid or a solid, as is the case of emulsion-style ointment bases and creams. Other means of stabilizing emulsions entail the use of emulsifiers that can be incorporated into either phase of the emulsion. Emulsifiers can broadly be classified into four categories: synthetic surfactants, naturally occurring emulsifiers, absorption bases, and finely dispersed solids (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Synthetic surfactants, also known as surface active agents, have found wide applicability in the formulation of emulsions and have been reviewed in the literature (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic and comprise a hydrophilic and a hydrophobic portion. The ratio of the hydrophilic to the hydrophobic nature of the surfactant has been termed the hydrophile/lipophile balance (HLB) and is a valuable tool in categorizing and selecting surfactants in the preparation of formulations. Surfactants can be classified into different classes based on the nature of the hydrophilic group: nonionic, anionic, cationic, and amphoteric (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y. Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285).

Naturally occurring emulsifiers used in emulsion formulations include lanolin, beeswax, phosphatides, lecithin, and acacia. Absorption bases possess hydrophilic properties such that they can soak up water to form w/o emulsions yet retain their semisolid consistencies, such as anhydrous lanolin and hydrophilic petrolatum. Finely divided solids have also been used as good emulsifiers especially in combination with surfactants and in viscous preparations. These include polar inorganic solids, such as heavy metal hydroxides, nonswelling clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate, and colloidal magnesium aluminum silicate, pigments and nonpolar solids such as carbon or glyceryl tristearate.

A large variety of non-emulsifying materials are also included in emulsion formulations and contribute to the properties of emulsions. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, humectants, hydrophilic colloids, preservatives, and antioxidants (Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Hydrophilic colloids or hydrocolloids include naturally occurring gums and synthetic polymers such as polysaccharides (for example, acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (for example, carboxymethylcellulose and carboxypropylcellulose), and synthetic polymers (for example, carbomers, cellulose ethers, and carboxyvinyl polymers). These disperse or swell in water to form colloidal solutions that stabilize emulsions by forming strong interfacial films around the dispersed-phase droplets and by increasing the viscosity of the external phase.

Since emulsions often contain a number of ingredients such as carbohydrates, proteins, sterols and phosphatides that can readily support the growth of microbes, these formulations often incorporate preservatives. Commonly used preservatives included in emulsion formulations include methyl paraben, propyl paraben, quaternary ammonium salts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boric acid. Antioxidants are also commonly added to emulsion formulations to prevent deterioration of the formulation. Antioxidants used can be free radical scavengers such as tocopherols, alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite, and antioxidant synergists such as citric acid, tartaric acid, and lecithin.

The application of emulsion formulations via dermatological, oral, and parenteral routes, and methods for their manufacture have been reviewed in the literature (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Emulsion formulations for oral delivery have been very widely used because of ease of formulation, as well as efficacy from an absorption and bioavailability standpoint (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil base laxatives, oil-soluble vitamins, and high fat nutritive preparations are among the materials that have commonly been administered orally as o/w emulsions.

ii. Microemulsions

In one embodiment of the present invention, the iRNAs are formulated as microemulsions. A microemulsion can be defined as a system of water, oil, and amphiphile which is a single optically isotropic and thermodynamically stable liquid solution (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Typically microemulsions are systems that are prepared by first dispersing an oil in an aqueous surfactant solution and then adding a sufficient amount of a fourth component, generally an intermediate chain-length alcohol to form a transparent system. Therefore, microemulsions have also been described as thermodynamically stable, isotropically clear dispersions of two immiscible liquids that are stabilized by interfacial films of surface-active molecules (Leung and Shah, in: Controlled Release of Drugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215). Microemulsions commonly are prepared via a combination of three to five components that include oil, water, surfactant, cosurfactant and electrolyte. Whether the microemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w) type is dependent on the properties of the oil and surfactant used and on the structure and geometric packing of the polar heads and hydrocarbon tails of the surfactant molecules (Schott, in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).

The phenomenological approach utilizing phase diagrams has been extensively studied and has yielded a comprehensive knowledge, to one skilled in the art, of how to formulate microemulsions (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared to conventional emulsions, microemulsions offer the advantage of solubilizing water-insoluble drugs in a formulation of thermodynamically stable droplets that are formed spontaneously.

Surfactants used in the preparation of microemulsions include, but are not limited to, ionic surfactants, non-ionic surfactants, Brij® 96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750), alone or in combination with cosurfactants. The cosurfactant, usually a short-chain alcohol such as ethanol, 1-propanol, and 1-butanol, serves to increase the interfacial fluidity by penetrating into the surfactant film and consequently creating a disordered film because of the void space generated among surfactant molecules. Microemulsions can, however, be prepared without the use of cosurfactants and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase can typically be, but is not limited to, water, an aqueous solution of the drug, glycerol, PEG300, PEG400, polyglycerols, propylene glycols, and derivatives of ethylene glycol. The oil phase can include, but is not limited to, materials such as Captex® 300, Captex® 355, Capmul® MCM, fatty acid esters, medium chain (C8-C12) mono, di, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils, and silicone oil.

Microemulsions are particularly of interest from the standpoint of drug solubilization and the enhanced absorption of drugs. Lipid based microemulsions (both o/w and w/o) have been proposed to enhance the oral bioavailability of drugs, including peptides (see e.g., U.S. Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp. Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages of improved drug solubilization, protection of drug from enzymatic hydrolysis, possible enhancement of drug absorption due to surfactant-induced alterations in membrane fluidity and permeability, ease of preparation, ease of oral administration over solid dosage forms, improved clinical potency, and decreased toxicity (see e.g., U.S. Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci., 1996, 85, 138-143). Often microemulsions can form spontaneously when their components are brought together at ambient temperature. This can be particularly advantageous when formulating thermolabile drugs, peptides or iRNAs. Microemulsions have also been effective in the transdermal delivery of active components in both cosmetic and pharmaceutical applications. It is expected that the microemulsion compositions and formulations of the present invention will facilitate the increased systemic absorption of iRNAs and nucleic acids from the gastrointestinal tract, as well as improve the local cellular uptake of iRNAs and nucleic acids.

Microemulsions of the present invention can also contain additional components and additives such as sorbitan monostearate (Grill® 3), Labrasol®, and penetration enhancers to improve the properties of the formulation and to enhance the absorption of the iRNAs and nucleic acids of the present invention. Penetration enhancers used in the microemulsions of the present invention can be classified as belonging to one of five broad categories—surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of these classes has been discussed above.

iii. Microparticles

An iRNA of the invention may be incorporated into a particle, e.g., a microparticle. Microparticles can be produced by spray-drying, but may also be produced by other methods including lyophilization, evaporation, fluid bed drying, vacuum drying, or a combination of these techniques.

iv. Penetration Enhancers

In one embodiment, the present invention employs various penetration enhancers to effect the efficient delivery of nucleic acids, particularly iRNAs, to the skin of animals. Most drugs are present in solution in both ionized and nonionized forms. However, usually only lipid soluble or lipophilic drugs readily cross cell membranes. It has been discovered that even non-lipophilic drugs can cross cell membranes if the membrane to be crossed is treated with a penetration enhancer. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also enhance the permeability of lipophilic drugs.

Penetration enhancers can be classified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Such compounds are well known in the art.

v. Carriers

Certain compositions of the present invention also incorporate carrier compounds in the formulation. As used herein, “carrier compound” or “carrier” can refer to a nucleic acid, or analog thereof, which is inert (i.e., does not possess biological activity per se) but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of a nucleic acid having biological activity by, for example, degrading the biologically active nucleic acid or promoting its removal from circulation. The coadministration of a nucleic acid and a carrier compound, typically with an excess of the latter substance, can result in a substantial reduction of the amount of nucleic acid recovered in the liver, kidney or other extracirculatory reservoirs, presumably due to competition between the carrier compound and the nucleic acid for a common receptor. For example, the recovery of a partially phosphorothioate dsRNA in hepatic tissue can be reduced when it is coadministered with polyinosinic acid, dextran sulfate, polycytidic acid or 4-acetamido-4′isothiocyano-stilbene-2,2′-disulfonic acid (Miyao et al., DsRNA Res. Dev., 1995, 5, 115-121; Takakura et al., DsRNA & Nucl. Acid Drug Dev., 1996, 6, 177-183.

vi. Excipients

In contrast to a carrier compound, a “pharmaceutical carrier” or “excipient” is a pharmaceutically acceptable solvent, suspending agent, or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal. The excipient can be liquid or solid and is selected, with the planned manner of administration in mind, so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition. Typical pharmaceutical carriers include, but are not limited to, binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycolate, etc.); and wetting agents (e.g., sodium lauryl sulphate, etc).

Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can also be used to formulate the compositions of the present invention. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone, and the like.

Formulations for topical administration of nucleic acids can include sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions of the nucleic acids in liquid or solid oil bases. The solutions can also contain buffers, diluents and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can be used.

Suitable pharmaceutically acceptable excipients include, but are not limited to, water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone, and the like.

vii. Other Components

The compositions of the present invention can additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels. Thus, for example, the compositions can contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or can contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.

Aqueous suspensions can contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol, or dextran. The suspension can also contain stabilizers.

In some embodiments, pharmaceutical compositions featured in the invention include (a) one or more iRNA and (b) one or more agents which function by a non-iRNA mechanism and which are useful in treating an IGFALS or IGF-1-associated disorder.

Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds that exhibit high therapeutic indices are preferred.

The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of compositions featured herein in the invention lies generally within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the methods featured in the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range of the compound or, when appropriate, of the polypeptide product of a target sequence (e.g., achieving a decreased concentration of the polypeptide) that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography.

In addition to their administration, as discussed above, the iRNAs featured in the invention can be administered in combination with other known agents effective in treatment of pathological processes mediated by IGFALS or IGF-1 expression. In any event, the administering physician can adjust the amount and timing of iRNA administration on the basis of results observed using standard measures of efficacy known in the art or described herein.

VI. Methods of the Invention

The present invention also provides methods of inhibiting expression of an IGFALS gene or IGF-1 gene in a cell. The methods include contacting a cell with an RNAi agent, e.g., double stranded RNAi agent, in an amount effective to inhibit expression of IGFALS or IGF-1 in the cell, thereby inhibiting expression of IGFALS or IGF-1 in the cell.

Contacting of a cell with an iRNA, e.g., a double stranded RNAi agent, may be done in vitro or in vivo. Contacting a cell in vivo with the iRNA includes contacting a cell or group of cells within a subject, e.g., a human subject, with the iRNA. Combinations of in vitro and in vivo methods of contacting a cell are also possible. Contacting a cell may be direct or indirect, as discussed above.

Furthermore, contacting a cell may be accomplished via a targeting ligand, including any ligand described herein or known in the art. In preferred embodiments, the targeting ligand is a carbohydrate moiety, e.g., a GalNAc₃ ligand, or any other ligand that directs the RNAi agent to a site of interest.

The term “inhibiting,” as used herein, is used interchangeably with “reducing,” “silencing,” “downregulating”, “suppressing”, and other similar terms, and includes any level of inhibition.

The phrase “inhibiting expression of an IGFALS or “inhibiting expression of an IGF-1” is intended to refer to inhibition of expression of any IGFALS gene or IGF-1 gene (such as, e.g., a mouse IGFALS gene or IGF-1 gene, a rat IGFALS gene or IGF-1 gene, a monkey IGFALS gene or IGF-1 gene, or a human IGFALS gene or IGF-1 gene) as well as variants or mutants of an IGFALS gene or IGF-1 gene. Thus, the IGFALS gene or IGF-1 gene may be a wild-type IGFALS gene or IGF-1 gene, a mutant IGFALS gene or IGF-1 gene (such as a mutant IGFALS gene or IGF-1 gene), or a transgenic IGFALS gene or IGF-1 gene in the context of a genetically manipulated cell, group of cells, or organism.

“Inhibiting expression of an IGFALS gene” or “inhibiting expression of an IGF-1 gene” includes any level of inhibition of an IGFALS gene or an IGF-1 gene, e.g., at least partial suppression of the expression of an IGFALS gene or an IGF-1 gene. The expression of the IGFALS gene or an IGF-1 gene may be assessed based on the level, or the change in the level, of any variable associated with IGFALS gene or an IGF-1 gene expression, e.g., IGFALS mRNA or IGF-1 mRNA level or an IGFALS protein level or an IGF-1 protein level. This level may be assessed in an individual cell or in a group of cells, including, for example, a sample derived from a subject.

Inhibition may be assessed by a decrease in an absolute or relative level of one or more variables that are associated with IGFALS or IGF-1 expression compared with a control level. The control level may be any type of control level that is utilized in the art, e.g., a pre-dose baseline level, or a level determined from a similar subject, cell, or sample that is untreated or treated with a control (such as, e.g., buffer only control or inactive agent control).

In some embodiments of the methods of the invention, expression of an IGFALS or IGF-1 gene is inhibited by at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or to below the level of detection of the assay. In some embodiments, the inhibition of expression of an IGFALS gene or an IGF-1 gene results in normalization of the level of IGF-1 such that the difference between the level before treatment and a normal control level is reduced by at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.

Inhibition of the expression of an IGFALS gene or an IGF-1 gene may be manifested by a reduction of the amount of mRNA expressed by a first cell or group of cells (such cells may be present, for example, in a sample derived from a subject) in which an IGFALS gene or an IGF-1 gene is transcribed and which has or have been treated (e.g., by contacting the cell or cells with an iRNA of the invention, or by administering an iRNA of the invention to a subject in which the cells are or were present) such that the expression of an IGFALS gene or an IGF-1 gene is inhibited, as compared to a second cell or group of cells substantially identical to the first cell or group of cells but which has not or have not been so treated (control cell(s) not treated with an iRNA or not treated with an iRNA targeted to the gene of interest). In preferred embodiments, the inhibition is assessed by the method provided in Example 2 and expressing the level of mRNA in treated cells as a percentage of the level of mRNA in control cells, using the following formula:

${\frac{\left( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{control}\mspace{14mu}{cells}} \right) - \left( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{treated}\mspace{14mu}{cells}} \right)}{\left( {{mRNA}\mspace{20mu}{in}\mspace{20mu}{control}\mspace{14mu}{cells}} \right)} \cdot 100}\%$

In other embodiments, inhibition of the expression of an IGFALS gene or an IGF-1 gene may be assessed in terms of a reduction of a parameter that is functionally linked to IGFALS or IGF-1 gene expression, e.g., IGFALS or IGF-1 protein expression or IGF signaling pathways. IGFALS or IGF-1 gene silencing may be determined in any cell expressing IGFALS or IGF-1, either endogenous or heterologous from an expression construct, and by any assay known in the art.

Inhibition of the expression of an IGFALS or IGF-1 protein may be manifested by a reduction in the level of the IGFALS or IGF-1 protein that is expressed by a cell or group of cells (e.g., the level of protein expressed in a sample derived from a subject). As explained above, for the assessment of mRNA suppression, the inhibition of protein expression levels in a treated cell or group of cells may similarly be expressed as a percentage of the level of protein in a control cell or group of cells.

A control cell or group of cells that may be used to assess the inhibition of the expression of an IGFALS or IGF-1 gene includes a cell or group of cells that has not yet been contacted with an RNAi agent of the invention. For example, the control cell or group of cells may be derived from an individual subject (e.g., a human or animal subject) prior to treatment of the subject with an RNAi agent.

In certain embodiments, inhibition of expression of an IGF-1 gene may be manifested in a reduction in the difference between a normal level of IGF-1 mRNA or protein and an abnormal level of IGF-1 mRNA or protein in a subject or in a specific tissue in the subject, e.g., mRNA in the liver of the subject or IGF-1 protein in subject serum. That is, inhibition may be manifested in a normalization of expression as compared to an appropriate control.

The level of IGFALS mRNA or IGF-1 mRNA that is expressed by a cell or group of cells, or the level of circulating IGFALS mRNA or IGF-1 mRNA, may be determined using any method known in the art for assessing mRNA expression. In one embodiment, the level of expression of IGFALS or IGF-1 in a sample is determined by detecting a transcribed polynucleotide, or portion thereof, e.g., mRNA of the IGFALS gene or IGF-1 gene. RNA may be extracted from cells using RNA extraction techniques including, for example, using acid phenol/guanidine isothiocyanate extraction (RNAzol B; Biogenesis), RNeasy™ RNA preparation kits (Qiagen®) or PAXgene (PreAnalytix, Switzerland). Typical assay formats utilizing ribonucleic acid hybridization include nuclear run-on assays, RT-PCR, RNase protection assays, northern blotting, in situ hybridization, and microarray analysis. Circulating IGFALS or IGF-1 mRNA may be detected using methods the described in PCT Publication WO2012/177906, the entire contents of which are hereby incorporated herein by reference.

In some embodiments, the level of expression of IGFALS or IGF-1 is determined using a nucleic acid probe. The term “probe”, as used herein, refers to any molecule that is capable of selectively binding to a specific IGFALS or IGF-1. Probes can be synthesized by one of skill in the art, or derived from appropriate biological preparations. Probes may be specifically designed to be labeled. Examples of molecules that can be utilized as probes include, but are not limited to, RNA, DNA, proteins, antibodies, and organic molecules.

Isolated mRNA can be used in hybridization or amplification assays that include, but are not limited to, Southern or northern analyses, polymerase chain reaction (PCR) analyses and probe arrays. One method for the determination of mRNA levels involves contacting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize to IGFALS mRNA or IGF-1 mRNA. In one embodiment, the mRNA is immobilized on a solid surface and contacted with a probe, for example by running the isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane, such as nitrocellulose. In an alternative embodiment, the probe(s) are immobilized on a solid surface and the mRNA is contacted with the probe(s), for example, in an Affymetrix gene chip array. A skilled artisan can readily adapt known mRNA detection methods for use in determining the level of IGFALS mRNA or IGF-1 mRNA.

An alternative method for determining the level of expression of IGFALS or IGF-1 in a sample involves the process of nucleic acid amplification and/or reverse transcriptase (to prepare cDNA) of for example mRNA in the sample, e.g., by RT-PCR (the experimental embodiment set forth in Mullis, 1987, U.S. Pat. No. 4,683,202), ligase chain reaction (Barany (1991) Proc. Natl. Acad. Sci. USA 88:189-193), self sustained sequence replication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplification system (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi et al. (1988) Bio Technology 6:1197), rolling circle replication (Lizardi et al., U.S. Pat. No. 5,854,033) or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers. In particular aspects of the invention, the level of expression of IGFALS or IGF-1 is determined by quantitative fluorogenic RT-PCR (i.e., the TaqMan™ System).

The expression levels of IGFALS or IGF-1 mRNA may be monitored using a membrane blot (such as used in hybridization analysis such as northern, Southern, dot, and the like), or microwells, sample tubes, gels, beads or fibers (or any solid support comprising bound nucleic acids). See U.S. Pat. Nos. 5,770,722, 5,874,219, 5,744,305, 5,677,195 and 5,445,934, which are incorporated herein by reference. The determination of IGFALS or IGF-1 expression level may also comprise using nucleic acid probes in solution.

In preferred embodiments, the level of mRNA expression is assessed using branched DNA (bDNA) assays or real time PCR (qPCR). The use of these methods is described and exemplified in the Examples presented herein.

The level of IGFALS or IGF-1 protein expression may be determined using any method known in the art for the measurement of protein levels. Such methods include, for example, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, fluid or gel precipitin reactions, absorption spectroscopy, a colorimetric assays, spectrophotometric assays, flow cytometry, immunodiffusion (single or double), immunoelectrophoresis, western blotting, radioimmunoassay (RIA), enzyme-linked immunosorbent assays (ELISAs), immunofluorescent assays, electrochemiluminescence assays, and the like.

In some embodiments, the efficacy of the methods of the invention in the treatment of an IGF system-associated disease is assessed by a decrease in IGFALS mRNA or IGF-1 mRNA level (by liver biopsy) or IGFALS or IGF-1 protein level, typically determined in serum.

In some embodiments, the efficacy of the methods of the invention in the treatment of acromegaly can be monitored by evaluating a subject for normalization of at least one sign or symptom of acromegaly previously displayed in the subject including, elevated IGF-1 level, sleep apnea, joint pain, symptomatic carpal tunnel syndrome, hypertension, biventricular cardiac hypertrophy, cardiac arrhythmia, fatigue, and weakness. These symptoms may be assessed in vitro or in vivo using any method known in the art. Although the nadir GH suppression after administration of glucose can be considered the “gold standard” test for acromegaly (Katznelson et al., 2011, Endocrine Practice), suppression may not be observed after treatment with the RNAi agents provided herein due to their proposed mechanism of action. Moreover, subjects may have accomplished clinically relevant beneficial outcomes with lowering of IGF-1 without reaching normal GH levels.

It is understood that normal IGF-1 levels are dependent both on the age and gender of the subject, with younger subjects having lower IGF-1 levels than older subjects. Therefore, when comparing IGF-1 levels to determine the lowering or normalizing of the level, an appropriate control must be selected. Appropriate controls include, for example, an IGF-1 level prior to treatment (when available) or an age and gender matched control. In certain embodiments, IGF-1 levels are monitored or tested on multiple occasions to confirm a change in IGF-1 level in a subject. In preferred embodiments, the IGF-1 level is decreased sufficiently to provide a clinically beneficial outcome for the subject.

In some embodiments, the efficacy of the method of the invention in treatment of cancer can be monitored by evaluating a subject for maintenance or preferably reduction of tumor burden of the primary tumor or metastatic tumor(s) or the prevention of metastasis. Methods for detection and monitoring of tumor burden are known in the art, e.g., RECIST criteria as provided in Eisenhauer et al., 2009, New response evaluation criteria in solid tumours: Revised RECIST guideline (version 1.1). Eur. J. Cancer. 45:228-247.

In some embodiments of the methods of the invention, the iRNA is administered to a subject such that the iRNA is delivered to a specific site within the subject. The inhibition of expression of IGFALS or IGF-1 may be assessed using measurements of the level or change in the level of IGFALS or IGF-1 mRNA or IGFALS or IGF-1 protein in a sample derived from fluid or tissue from the specific site within the subject.

As used herein, the terms detecting or determining a level of an analyte are understood to mean performing the steps to determine if a material, e.g., protein, RNA, is present. As used herein, methods of detecting or determining include detection or determination of an anlyte level that is below the level of detection for the method used.

VII. Methods of Treating or Preventing IGF System-Associated Diseases

The present invention also provides methods of using an iRNA of the invention or a composition containing an iRNA of the invention to reduce or inhibit IGFALS or IGF-1 expression in a cell. The methods include contacting the cell with a dsRNA of the invention and maintaining the cell for a time sufficient to obtain degradation of the mRNA transcript of an IGFALS gene or an IGF-1 gene, thereby inhibiting expression of the IGFALS gene or an IGF-1 gene in the cell. Reduction in gene expression can be assessed by any methods known in the art. For example, a reduction in the expression of IGFALS or IGF-1 may be determined by determining the mRNA expression level of IGFALS or IGF-1, e.g., in a liver sample, using methods routine to one of ordinary skill in the art, e.g., northern blotting, qRT-PCR; by determining the protein level of IGFALS or IGF-1 using methods routine to one of ordinary skill in the art, such as western blotting, immunological techniques. A reduction in the expression of IGFALS or IGF-1 may also be assessed indirectly by measuring a decrease in biological activity of IGFALS or IGF-1 or measuring the level of IGF-1 in a subject sample (e.g., a serum sample).

In the methods of the invention the cell may be contacted in vitro or in vivo, i.e., the cell may be within a subject.

A cell suitable for treatment using the methods of the invention may be any cell that expresses an IGFALS or IGF-1 gene, typically a liver cell. A cell suitable for use in the methods of the invention may be a mammalian cell, e.g., a primate cell (such as a human cell or a non-human primate cell, e.g., a monkey cell or a chimpanzee cell), a non-primate cell (such as a cow cell, a pig cell, a camel cell, a llama cell, a horse cell, a goat cell, a rabbit cell, a sheep cell, a hamster, a guinea pig cell, a cat cell, a dog cell, a rat cell, a mouse cell, a lion cell, a tiger cell, a bear cell, or a buffalo cell), a bird cell (e.g., a duck cell or a goose cell), or a whale cell. In one embodiment, the cell is a human cell, e.g., a human liver cell.

IGFALS expression or IGF-1 expression is inhibited in the cell by at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or to a level below the level of detection of the assay. IGFALS expression or IGF-1 expression is inhibited in the cell such that the difference between the level of expression in a subject with an IGF system-associated disease and the normal level of expression is reduce by at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.

The in vivo methods of the invention may include administering to a subject a composition containing an iRNA, where the iRNA includes a nucleotide sequence that is complementary to at least a part of an RNA transcript of the IGFALS gene or IGF-1 gene of the mammal to be treated. When the organism to be treated is a mammal such as a human, the composition can be administered by any means known in the art including, but not limited to oral, intraperitoneal, or parenteral routes, including intracranial (e.g., intraventricular, intraparenchymal, and intrathecal), intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), nasal, rectal, and topical (including buccal and sublingual) administration. In certain embodiments, the compositions are administered by intravenous infusion or injection. In certain embodiments, the compositions are administered by subcutaneous injection.

In some embodiments, the administration is via a depot injection. A depot injection may release the iRNA in a consistent way over a prolonged time period. Thus, a depot injection may reduce the frequency of dosing needed to obtain a desired effect, e.g., a desired inhibition of IGFALS or IGF-1, or a therapeutic or prophylactic effect. A depot injection may also provide more consistent serum concentrations. Depot injections may include subcutaneous injections or intramuscular injections. In preferred embodiments, the depot injection is a subcutaneous injection.

In some embodiments, the administration is via a pump. The pump may be an external pump or a surgically implanted pump. In certain embodiments, the pump is a subcutaneously implanted osmotic pump. In other embodiments, the pump is an infusion pump. An infusion pump may be used for intravenous, subcutaneous, arterial, or epidural infusions. In preferred embodiments, the infusion pump is a subcutaneous infusion pump. In other embodiments, the pump is a surgically implanted pump that delivers the iRNA to the liver.

The mode of administration may be chosen based upon whether local or systemic treatment is desired and based upon the area to be treated. The route and site of administration may be chosen to enhance targeting.

In one aspect, the present invention also provides methods for inhibiting the expression of an IGFALS or IGF-1 gene in a mammal. The methods include administering to the mammal a composition comprising a dsRNA that targets an IGFALS or an IGF-1 gene in a cell of the mammal and maintaining the mammal for a time sufficient to obtain degradation of the mRNA transcript of the IGFALS gene or the IGF-1 gene, thereby inhibiting expression of the IGFALS gene or the IGF-1 gene in the cell. Reduction in gene expression can be assessed by any methods known it the art and by methods, e.g. qRT-PCR, described herein. Reduction in protein production can be assessed by any methods known it the art and by methods, e.g. ELISA, described herein. In one embodiment, a puncture liver biopsy sample serves as the tissue material for monitoring the reduction in the IGFALS gene or the IGF-1 gene or protein expression.

The present invention further provides methods of treatment of a subject in need thereof. The treatment methods of the invention include administering an iRNA of the invention to a subject, e.g., a subject that would benefit from a reduction or inhibition of IGFALS or IGF-1 expression, in a therapeutically effective amount of an iRNA targeting an IGFALS gene or an IGF-1 gene or a pharmaceutical composition comprising an iRNA targeting an IGFALS gene or an IGF-1 gene.

An iRNA of the invention may be administered as a “free iRNA.” A free iRNA is administered in the absence of a pharmaceutical composition. The naked iRNA may be in a suitable buffer solution. The buffer solution may comprise acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof. In one embodiment, the buffer solution is phosphate buffered saline (PBS). The pH and osmolarity of the buffer solution containing the iRNA can be adjusted such that it is suitable for administering to a subject.

Alternatively, an iRNA of the invention may be administered as a pharmaceutical composition, such as a dsRNA liposomal formulation.

Subjects that would benefit from a reduction or inhibition of IGFALS gene or an IGF-1 gene expression are those having a disorder of elevated growth hormone, e.g., acromegaly, or a disorder of elevated insulin signaling, e.g., cancer. In another embodiment, a subject having a disorder of elevated growth hormone has one or more signs or symptoms associated with acromegaly or elevated growth hormone including, but not limited to, elevated IGF-1 level, somatic enlargement (soft tissue and bony overgrowth), excessive sweating, jaw overgrowth, sleep apnea, osteoarthropathy, joint pain, symptomatic carpal tunnel syndrome, hypertension, biventricular cardiac hypertrophy, cardiac arrhythmia, fatigue, weakness, diabetes mellitus, menstrual irregularities in women and sexual dysfunction in men, headache, and visual field loss (attributable to optic chiasmal compression) and diplopia (due to cranial nerve palsy); in conjunction with an elevated growth hormone level.

Treatment of a subject that would benefit from a reduction or inhibition of IGFALS or IGF-1 gene expression and normalization of growth hormone levels includes therapeutic treatment (e.g., of a subject is suffering from acromegaly) and prophylactic treatment (e.g., of a subject does not meet the diagnostic criteria of acromegaly or may have elevated or fluctuating growth hormone, or IGFALS, or IGF-1 levels, or a subject may be at risk of developing acromegaly). Treatment of a subject that would benefit from a reduction or inhibition of IGFALS gene expression or IGF-1 gene expression can also include treatment of cancer.

The invention further provides methods for the use of an iRNA or a pharmaceutical composition thereof, e.g., for treating a subject that would benefit from reduction or inhibition of IGFALS or IGF-1 expression, e.g., a subject having a disorder of elevated growth hormone, in combination with other pharmaceuticals or other therapeutic methods, e.g., with known pharmaceuticals or known therapeutic methods, such as, for example, those which are currently employed for treating these disorders. For example, in certain embodiments, an iRNA targeting IGFALS or IGF-1 is administered in combination with an agent useful in treating a disorder of elevated growth hormone as described elsewhere herein.

The invention provides methods for the treatment of cancer, e.g., IGF-1 dependent cancer, IGF-1 receptor positive cancer, or metastatic or potentially metastatic cancer. In certain embodiments, the iRNAs of the invention are used in conjunction with various standards of treatment of cancer, e.g., chemotherapeutic agents, surgery, radiation; and combinations thereof.

The iRNA and additional therapeutic agents may be administered at the same time or in the same combination, e.g., parenterally, or the additional therapeutic agent can be administered as part of a separate composition or at separate times or by another method known in the art or described herein.

In one embodiment, the method includes administering a composition featured herein such that expression of the target IGFALS gene or IGF-1 gene is decreased, such as for about 1, 2, 3, 4, 5, 6, 7, 8, 12, 16, 18, 24 hours, 28, 32, or about 36 hours. In one embodiment, expression of the target IGFALS gene or IGF-1 gene is decreased for an extended duration, e.g., at least about two, three, four days or more, e.g., about one week, two weeks, three weeks, or four weeks or longer.

Preferably, the iRNAs useful for the methods and compositions featured herein specifically target RNAs (primary or processed) of the target IGFALS gene or IGF-1 gene. Compositions and methods for inhibiting the expression of these genes using iRNAs can be prepared and performed as described herein.

Administration of the iRNA according to the methods of the invention may result in a reduction of the severity, signs, symptoms, or markers of such diseases or disorders in a patient with a disorder of elevated growth hormone, elevated IGFALS, elevated IGF-1, or an IGF-1 responsive tumor. By “reduction” in this context is meant a statistically significant decrease in such level. The reduction can be, for example, at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or to below the level of detection of the assay used.

Efficacy of treatment or prevention of disease can be assessed, for example by measuring disease progression, disease remission, symptom severity, reduction in pain, quality of life, dose of a medication required to sustain a treatment effect, level of a disease marker, or any other measurable parameter appropriate for a given disease being treated or targeted for prevention. It is well within the ability of one skilled in the art to monitor efficacy of treatment or prevention by measuring any one of such parameters, or any combination of parameters. For example, efficacy of treatment of a disorder of IGF signaling may be assessed, for example, by periodic monitoring of IGF-1 or IGFALS levels, e.g., serum IGF-1 or IGFALS levels. For subjects suffering from acromegaly a decrease in one or more signs or symptoms including, but not limited to sleep apnea, joint pain, symptomatic carpal tunnel syndrome, hypertension, biventricular cardiac hypertrophy, cardiac arrhythmia, fatigue, and weakness can be an indication of treatment of acromegaly. Similarly a delay or lessening of the severity of the co-morbidities associated with acromegaly such as hypertension, hypertrophy, stroke, diabetes, and sleep apnea can demonstrate efficacy of treatment.

Efficacy of treatment of cancer can be demonstrated by stabilization or a decrease in tumor burden as demonstrated by a stabilization or decrease in tumor burden of the primary tumor, metastatic tumors, or the delay or prevention of tumor metastasis. Diagnostic and monitoring methods are known in the art and are also provided herein.

Comparisons of the later readings with the initial readings provide a physician an indication of whether the treatment is effective. It is well within the ability of one skilled in the art to monitor efficacy of treatment or prevention by measuring any one of such parameters, or any combination of parameters. In connection with the administration of an iRNA targeting IGFALS or IGF-1, or pharmaceutical composition thereof, “effective against” an IGF system-associated disorder indicates that administration in a clinically appropriate manner results in a beneficial effect for at least a statistically significant fraction of patients, such as a improvement of symptoms, a cure, a reduction in disease, extension of life, improvement in quality of life, or other effect generally recognized as positive by medical doctors familiar with treating IGF system-associated disorders.

A treatment or preventive effect is evident when there is a statistically significant improvement in one or more parameters of disease status, or by a failure to worsen or to develop symptoms where they would otherwise be anticipated. As an example, a favorable change of at least 10% in a measurable parameter of disease, and preferably at least 20%, 30%, 40%, 50% or more can be indicative of effective treatment. Efficacy for a given iRNA drug or formulation of that drug can also be judged using an experimental animal model for the given disease as known in the art. When using an experimental animal model, efficacy of treatment is evidenced when a statistically significant reduction in a marker or symptom is observed.

Alternatively, the efficacy can be measured by a reduction in the severity of disease as determined by one skilled in the art of diagnosis based on a clinically accepted disease severity grading scale. Any positive change resulting in e.g., lessening of severity of disease measured using the appropriate scale, represents adequate treatment using an iRNA or iRNA formulation as described herein.

Subjects can be administered a therapeutic amount of iRNA, such as about 0.01 mg/kg to about 200 mg/kg.

The iRNA can be administered by intravenous infusion over a period of time, on a regular basis. In certain embodiments, after an initial treatment regimen, the treatments can be administered on a less frequent basis. Administration of the iRNA can reduce IGFALS or IGF-1 levels, e.g., in a cell, tissue, blood, urine, or other compartment of the patient by at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or below the level of detection of the assay method used. It is noted that a reduction in IGFALS will not likely result in a decrease in growth hormone levels in a subject with acromegaly. Administration of the iRNA can reduce the difference in the subject IGF-1 levels and a normal IGF-1 level, e.g., in a cell, tissue, blood, urine, or other compartment of the patient by at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.

Before administration of a full dose of the iRNA, patients can be administered a smaller dose, such as a 5% infusion reaction, and monitored for adverse effects, such as an allergic reaction. In another example, the patient can be monitored for unwanted immunostimulatory effects, such as increased cytokine (e.g., TNF-alpha or INF-alpha) levels.

Alternatively, the iRNA can be administered subcutaneously, i.e., by subcutaneous injection. One or more injections may be used to deliver the desired daily dose of iRNA to a subject. The injections may be repeated over a period of time. The administration may be repeated on a regular basis. In certain embodiments, after an initial treatment regimen, the treatments can be administered on a less frequent basis. A repeat-dose regimen may include administration of a therapeutic amount of iRNA on a regular basis, such as every other day or to once a year. In certain embodiments, the iRNA is administered about once per month to about once per quarter (i.e., about once every three months).

IX. Diagnostic Criteria and Treatment for Acromegaly

Diagnostic criteria for acromegaly are set forth in the American Association of Clinical Endocrinologists Medical Guidelines for Clinical Practice for the Diagnosis and Treatment of Acromegaly—2011 Update (Katznelson et al., Endocr. Pract. 17(Suppl. 4), incorporated herein by reference. Further details and citations can be found therein.

Acromegaly is a clinical syndrome that, depending on its stage of progression, may not manifest with clear diagnostic features. Diagnosis should be considered in patients with 2 or more of the following comorbidities: new-onset diabetes, diffuse arthralgias, new-onset or difficult-to-control hypertension, cardiac disease including biventricular hypertrophy and diastolic or systolic dysfunction, fatigue, headaches, carpal tunnel syndrome, sleep apnea syndrome, diaphoresis, loss of vision, colon polyps, and progressive jaw malocclusion. A serum IGF-I level, if accompanied by a large number of results from age- and sex-matched normal subjects, is a good tool to assess integrated GH secretion and is excellent for diagnosis, monitoring, and especially screening. A random IGF-I value (a marker of integrated GH secretion) should be measured for diagnosis and for monitoring after a therapeutic intervention. Serum GH assays are not standardized and should not be used interchangably. The nadir GH suppression after administration of glucose has been considered the “gold standard” test for acromegaly, however, a conflict exists regarding the threshold for diagnosis. The panel recommends that GH measurements be performed at baseline, then every 30 minutes for a total of 120 minutes after administration of glucose. The inability to suppress serum GH to less than 1 ng/mL after glucose administration is typically considered the diagnostic criterion for acromegaly, however, in a consensus guideline in 2000, the diagnosis of acromegaly was excluded if the patient had a random GH measurement less than 0.4 ng/mL and a normal IGF-I value. Although a nadir GH concentration of less than 1 ng/mL after administration of glucose is the standard recommendation for a normal response, the 2011 panel suggests consideration of a lower nadir GH cut point at 0.4 ng/mL after glucose administration because of the enhanced assay sensitivity and more frequent finding of modest GH hypersecretion. A diagnosis of acromegaly will be made by one of skill in the art considering the totality of the evidence for the patient under consideration.

Once a biochemical diagnosis of acromegaly has been made, a magnetic resonance imaging (MRI) scan of the pituitary gland should be performed because a pituitary GH-secreting adenoma is the most common cause of acromegaly. Visual field testing should be performed if there is optic chiasmal compression noted on the MRI or if the patient has complaints of reduced peripheral vision. Further biochemical testing should include a serum prolactin level (to evaluate for hyperprolactinemia) and assessment of anterior and posterior pituitary function (for potential hypopituitarism).

The goals of therapy for acromegaly are to (1) control biochemical indices of activity, (2) control tumor size and prevent local mass effects, (3) reduce signs and symptoms of disease, (4) prevent or improve medical comorbidities, and (5) prevent early mortality. The primary mode of therapy is surgery, which is recommended for all patients with microadenomas and for all patients who have macroadenomas with associated mass effects. In patients with macroadenomas without mass effects, and with low likelihood of surgical cure, a role for surgical de-bulking of macroadenomas to improve the response to subsequent medical therapy has been advocated, as well as primary medical therapy alone. Medical therapy is generally used in the adjuvant setting. Irradiation, either conventional fractionated RT or stereotactic radiosurgery, is largely relegated to an adjuvant role. Availability of specific therapeutic options and cost of these interventions are taken into account with decisions regarding therapy.

The goal of surgical interventions is to decrease tumor volume, thereby decreasing production of excess growth hormone and decompress the mass effect of macroadenomas on any normal remaining pituitary gland tissues, optic nerve, or surrounding critical structures. Surgical interventions can be curative for many subjects. Surgically resected tissue should be analyzed to understand the tumor biology to potentially provide guidance for treatment. Biochemical analyses are also performed post-operatively to assess the surgical outcome.

Medical therapy is used in conjunction with surgery. Studies have provided conflicting results regarding the benefits of treatment with medical interventions prior to surgery to change the nature of the tumor. The iRNAs provided herein can be used at any time in conjunction with surgical intervention (i.e., before or after surgery).

Adjunctive medical therapy is used in patients who cannot achieve a complete cure by surgical intervention. Medical therapies fall into three categories: dopamine agonists, somatostatin analogs (SSAs), and a GH receptor antagonist. Each of the medical interventions presents different risks and benefits, including substantial costs of some of the therapies.

Dopamine agonists include cabergoline and bromocriptine. The agents are a good first line therapy, especially in patients with mild biochemical activity, as they are relatively inexpensive and orally administered. However, side effects include gastrointestinal upset, orthostatic hypotension, headache, and nasal congestion.

Somatostatin analogs (SSAs) include octreotide (Sandostatin®) LAR (long-acting release, administered as an intramuscular injection) and lanreotide (Somatuline®) Autogel (administered as a deep subcutaneous depot injection). SSAs are less convenient for use than dopamine agonists as they must be administered by injection (50 mcg three times daily Sandostatin® Injection subcutaneously for 2 weeks followed by Sandostatin® LAR 20 mg intragluteally every 4 weeks for 3 months; or 60, 90, or 120 mg of Somatuline® every 28 days by deep subcutaneous injection). SSAs are effective in normalizing IGF-I and GH levels in approximately 55% of patients. The clinical and biochemical responses to SSAs are inversely related to tumor size and degree of GH hypersecretion. Octreotide LAR and lanreotide Autogel have similar efficacy profiles. In patients with an inadequate response to SSAs, the addition of cabergoline or pegvisomant (Somavert®) may be effective for further lowering one or both of GH and IGF-1 levels. Potential side effects of SSAs, include gastrointestinal upset, malabsorption, constipation, gallbladder disease, hair loss, and bradycardia.

Pegvisomant, a GH receptor antagonist, is administered by daily subcutaneous injection. Side effects of pegvisomant, include flulike illness, allergic reactions, and increase in liver enzymes. Patients treated with pegvisomant must undergo routine liver enzyme tests. Because endogenous GH levels increase with pegvisomant administration and pegvisomant may be cross-measured in GH assays, serum GH levels are not specific and should not be monitored in patients receiving pegvisomant. Instead, serum IGF-1 levels are monitored.

Combinations of various medical therapies may be useful in the treatment of some acromegaly patients.

Radiation therapy is used as an adjunctive treatment is patients who do not respond sufficiently to surgical or medical interventions.

Similar treatment strategies are used in children with gigantism, a type of acromegaly, which refers to excess GH secretion that occurs during childhood when the growth plates are open, leading to accelerated vertical growth.

Some of the comorbidities of acromegaly resolve upon decreasing the level of GH or decreasing the responsiveness of the subject to GH. However, others are not. Unlike soft tissue changes, bone enlargement is not reversible. Surgical interventions (e.g., carpal tunnel release, joint replacement surgery), physical therapy, and analgesic medications can be used to treat conditions associated with bone or soft tissue overgrowth. Respiratory disorders including sleep apnea and higher susceptibility to respiratory infections can be treated with standard interventions and preventive strategies (e.g., influenza and pneumococcal vaccinations). Cardiovascular disease, hypertension, and stroke can be managed using standard monitoring (e.g., blood pressure, cholesterol, and lipid level monitoring) and medical treatment. Subjects should be monitored for the development of type 2 diabetes and neoplasia, particularly colon polyps and neoplasia. Subjects should also be monitored for psychological complications related to the physical changes and deformities that can occur with the disease. As used herein, treatment can include, but does not require, resolution of the co-morbidities of acromegaly. Treatment can include, but does not require, prevention or reduction of the development of one or more of the comorbidities associated with acromegaly. As used herein, treatment for acromegaly can further include, but does not require, treatment of one or more of the comorbidities associated with acromegaly.

IX. Response Evaluation Criteria and Treatment of Cancer

Methods for detection of tumors and assessment of tumor burden are well known in the art. For example, the Response Evaluation Criteria in Solid Tumors (RECIST) guidelines were revised in 2008 and are fully set forth in Eisenhauer et al., 2009, New response evaluation criteria in solid tumours: Revised RECIST guideline (version 1.1). Eur. J. Cancer. 45:228-247. These guidelines can be used to determine if a subject has tumor regression or no tumor progression as demonstrated by a complete response (CR) or partial response (PR), or stable disease (SD), respectively, as provided therein for at least a sufficient time that the CR, PR, or SD is detected meets the threshold of treatment or effective treatment as provided herein. A subject with only progressive disease (PD) after administration of an iRNA provided herein is not considered to have a favorable response to or be effectively treated by the iRNA. The development of PD after a period of CR, PR, or PD is understood as having been effectively treated by the iRNA provided herein.

It is understood that the iRNA agents provided herein can be used in conjunction with other interventions for the treatment of cancer, e.g., surgery, chemotherapy, or radiation.

This invention is further illustrated by the following examples which should not be construed as limiting. The entire contents of all references, patents and published patent applications cited throughout this application, as well as the Figure and Sequence Listing, are hereby incorporated herein by reference.

EXAMPLES Example 1. iRNA Synthesis Source of Reagents

Where the source of a reagent is not specifically given herein, such reagent can be obtained from any supplier of reagents for molecular biology at a quality/purity standard for application in molecular biology.

IGFALS Transcripts and siRNA Design

A set of siRNAs targeting the human IGFALS, “insulin-like growth factor binding protein, acid labile subunit”, (human: NCBI refseqID NM_004970; NCBI GeneID: 3483), as well as toxicology-species IGFALS orthologs (cynomolgus monkey: XM_005590898; mouse: NM_008340; rat, NM_053329) were designed using custom Rand Python scripts. The human NM_004970 REFSEQ mRNA has a length of 2168 bases.

The rationale and method for the set of siRNA designs is as follows: the predicted efficacy for every potential 19mer siRNA from position 50 through position 2160 (the coding region and 3′ UTR) was determined with a linear model derived the direct measure of mRNA knockdown from more than 20,000 distinct siRNA designs targeting a large number of vertebrate genes. Subsets of the IGFALS siRNAs were designed with perfect or near-perfect matches between human, cynomolgus and rhesus monkey. A further subset was designed with perfect or near-perfect matches to mouse and rat IGFALS orthologs. For each strand of the siRNA, a custom Python script was used in a brute force search to measure the number and positions of mismatches between the siRNA and all potential alignments in the target species transcriptome. Extra weight was given to mismatches in the seed region, defined here as positions 2-9 of the antisense oligonucleotide, as well the cleavage site of the siRNA, defined here as positions 10-11 of the antisense oligonucleotide. The relative weight of the mismatches was 2.8; 1.2:1 for seed mismatches, cleavage site, and other positions up through antisense position 19. Mismatches in the first position were ignored. A specificity score was calculated for each strand by summing the value of each weighted mismatch. Preference was given to siRNAs whose antisense score in human and cynomolgus monkey was >=2.0 and predicted efficacy was >=50% knockdown of the IGFALS transcript.

A detailed list of the unmodified IGFALS sense and antisense strand sequences is shown in Tables 3, 6, and 12.

A detailed list of the modified IGFALS sense and antisense strand sequences is shown in Tables 5, 8, and 14.

IGF-I Transcripts and siRNA Design

A set of siRNAs targeting the human insulin like growth factor 1, “IGF1” (human: e.g., NCBI refseqID NM_000618; NCBI GeneID: 3479), as well as toxicology-species IGF1 orthologs (cynomolgus monkey: e.g., XM_005572039; mouse: e.g., NM_010512; rat, e.g., NM_178866) were designed using custom R and Python scripts. The human NM_00618 REFSEQ mRNA has a length of 7366 bases.

The rationale and method for the set of siRNA designs is as follows: the predicted efficacy for every potential 19mer siRNA from position 265 through position 7366 (the coding region and 3′ UTR) was determined with a linear model derived the direct measure of mRNA knockdown from more than 20,000 distinct siRNA designs targeting a large number of vertebrate genes. Subsets of the IGF1 siRNAs were designed with perfect or near-perfect matches between human and cynomolgus monkey. A further subset was designed with perfect or near-perfect matches to human, cynomolgus monkey and mouse IGF1 orthologs. A further subset was designed with perfect or near-perfect matches to mouse and rat IGF1 orthologs. For each strand of the siRNA, a custom Python script was used in a brute force search to measure the number and positions of mismatches between the siRNA and all potential alignments in the target species transcriptome. Extra weight was given to mismatches in the seed region, defined here as positions 2-9 of the antisense oligonucleotide, as well the cleavage site of the siRNA, defined here as positions 10-11 of the antisense oligonucleotide. The relative weight of the mismatches was 2.8; 1.2:1 for seed mismatches, cleavage site, and other positions up through antisense position 19. Mismatches in the first position were ignored. A specificity score was calculated for each strand by summing the value of each weighted mismatch. Preference was given to siRNAs whose antisense score in human and cynomolgus monkey was >=3.0 and predicted efficacy was >=70% knockdown of the human IGF1 transcripts.

A detailed list of the unmodified IGF-1 sense and antisense strand sequences is shown in Tables 9, 15, and 18.

A detailed list of the modified IGF-1 sense and antisense strand sequences is shown in Tables 11, 17, and 20.

siRNA Synthesis

siRNA sequences were synthesized at 1 μmol scale on a Mermade 192 synthesizer (BioAutomation) using the solid support mediated phosphoramidite chemistry. The solid support was controlled pore glass (500 A) loaded with custom GalNAc ligand or universal solid support (AM biochemical). Ancillary synthesis reagents, 2′-F and 2′-O-Methyl RNA and deoxy phosphoramidites were obtained from Thermo-Fisher (Milwaukee, Wis.) and Hongene (China). 2′F 2′-O-Methyl, GNA (glycol nucleic acids), 5′phosphate and other modifications are introduced using the corresponding phosphoramidites. Synthesis of 3′ GalNAc conjugated single strands was performed on a GalNAc modified CPG support. Custom CPG universal solid support was used for the synthesis of antisense single strands. Coupling time for all phosphoramidites (100 mM in acetonitrile) is 5 min employing 5-Ethylthio-1H-tetrazole (ETT) as activator (0.6 M in acetonitrile). Phosphorothioate linkages were generated using a 50 mM solution of 3-((Dimethylamino-methylidene) amino)-3H-1,2,4-dithiazole-3-thione (DDTT, obtained from Chemgenes (Wilmington, Mass., USA)) in anhydrous acetonitrile/pyridine (1:1 v/v). Oxidation time was 3 minutes. All sequences were synthesized with final removal of the DMT group (“DMT off”).

Upon completion of the solid phase synthesis, oligoribonucleotides were cleaved from the solid support and deprotected in sealed 96 deep well plates using 200 μL Aqueous Methylamine reagents at 60° C. for 20 minutes. For sequences containing 2′ ribo residues (2′-OH) that are protected with a tert-butyl dimethyl silyl (TBDMS) group, a second step deprotection is performed using TEA.3HF (triethylamine trihydro fluoride) reagent. To the methylamine deprotection solution, 200 uL of dimethyl sulfoxide (DMSO) and 300 ul TEA.3HF reagent were added and the solution was incubated for additional 20 min at 60° C. At the end of cleavage and deprotection step, the synthesis plate was allowed to come to room temperature and is precipitated by addition of 1 mL of acetontile: ethanol mixture (9:1). The plates are cooled at −80 C for 2 hours, supernatant was decanted carefully with the aid of a multi channel pipette. The oligonucleotide pellet was re-suspended in 20 mM NaOAc buffer and is desalted using a 5 mL HiTrap size exclusion column (GE Healthcare) on an AKTA Purifier System equipped with an A905 autosampler and a Frac 950 fraction collector. Desalted samples are collected in 96-well plates. Samples from each sequence were analyzed by LC-MS to confirm the identity, UV (260 nm) for quantification and a selected set of samples by IEX chromatography to determine purity.

Annealing of single strands was performed on a Tecan liquid handling robot. Equimolar mixture of sense and antisense single strands were combined and annealed in 96 well plates. After combining the complementary single strands, the 96-well plate was sealed tightly and heated in an oven at 100° C. for 10 minutes and allowed to come slowly to room temperature over a period 2-3 hours. The concentration of each duplex was normalized to 10 μM in 1×PBS.

Example 2—In Vitro Screening Cell Culture and Transfections

Hep3B (ATCC) were transfected by adding 4.9 μl of Opti-MEM plus 0.1 μl of Lipofectamine RNAiMax per well (Invitrogen, Carlsbad Calif. cat #13778-150) to 5 μl of siRNA duplexes per well into a 384-well plate and incubated at room temperature for 15 minutes. Firty μl of DMEM containing ˜5×10³ cells were then added to the siRNA mixture. Cells were incubated for 24 hours prior to RNA purification. Single dose experiments were performed at 10 nM and 0.1 nM and in some cases 1 nM final duplex concentration.

Total RNA Isolation Using DYNABEADS mRNA Isolation Kit

RNA was isolated using an automated protocol on a BioTek-EL406 platform using DYNABEADs (Invitrogen, cat #61012). Briefly, 50 μl of Lysis/Binding Buffer and 25 μl of lysis buffer containing 3 μl of magnetic beads were added to the plate with cells. Plates were incubated on an electromagnetic shaker for 10 minutes at room temperature and then magnetic beads were captured and the supernatant was removed. Bead-bound RNA was then washed 2 times with 150p Wash Buffer A and once with Wash Buffer B. Beads were then washed with 150 μl Elution Buffer, re-captured and supernatant removed.

cDNA Synthesis Using ABI High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, Calif., Cat #4368813)

Ten μl of a master mix containing 1 μl 10× Buffer, 0.4 μl 25×dNTPs, 1 μl 10× Random primers, 0.5 μl Reverse Transcriptase, 0.5 μl RNase inhibitor and 6.6 μl of H₂O per reaction was added to RNA isolated above. Plates were sealed, mixed, and incubated on an electromagnetic shaker for 10 minutes at room temperature, followed by 2 hours 37° C. Plates were then incubated at 81° C. for 8 minutes.

Real Time PCR

Two μl of cDNA were added to a master mix containing 0.5 μl of GAPDH TaqMan Probe (Hs99999905 ml), 0.5 μl IGFALS probe (HS00744047 Si) and 5 μl Lightcycler 480 probe master mix (Roche Cat #04887301001) per well in a 384 well plates (Roche cat #04887301001). Real time PCR was done in a LightCycler480 Real Time PCR system (Roche). Each duplex was tested at least two times and data were normalized to naïve cells or cells transfected with a non-targeting control siRNA.

To calculate relative fold change, real time data were analyzed using the ΔΔCt method and normalized to assays performed with cells transfected with 10 nM AD-1955, or mock transfected cells.

TABLE 2 Abbreviations of nucleotide monomers used in nucleic acid sequence representation. It will be understood that these monomers, when present in an oligonucleotide, are mutually linked by 5′-3′-phosphodiester bonds. Abbreviation Nucleotide(s) A Adenosine-3′-phosphate Af 2′-fluoroadenosine-3′-phosphate Afs 2′-fluoroadenosine-3′-phosphorothioate As adenosine-3′-phosphorothioate C cytidine-3′-phosphate Cf 2′-fluorocytidine-3′-phosphate Cfs 2′-fluorocytidine-3′-phosphorothioate Cs cytidine-3′-phosphorothioate G guanosine-3′-phosphate Gf 2′-fluoroguanosine-3′-phosphate Gfs 2′-fluoroguanosine-3′-phosphorothioate Gs guanosine-3′-phosphorothioate T 5′-methyluridine-3′-phosphate Tf 2′-fluoro-5-methyluridine-3′-phosphate Tfs 2′-fluoro-5-methyluridine-3′-phosphorothioate Ts 5-methyluridine-3′-phosphorothioate U Uridine-3′-phosphate Uf 2′-fluorouridine-3′-phosphate Ufs 2′-fluorouridine-3′-phosphorothioate Us uridine-3′-phosphorothioate N any nucleotide (G, A, C, T or U) a 2′-O-methyladenosine-3′-phosphate as 2′-O-methyladenosine-3′-phosphorothioate c 2′-O-methylcytidine-3′-phosphate cs 2′-O-methylcytidine-3′-phosphorothioate g 2′-O-methylguanosine-3′-phosphate gs 2′-O-methylguanosine-3′-phosphorothioate t 2′-O-methyl-5-methyluridine-3′-phosphate ts 2′-O-methyl-5-methyluridine-3′-phosphorothioate u 2′-O-methyluridine-3′-phosphate us 2′-O-methyluridine-3′-phosphorothioate s phosphorothioate linkage L96 N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol Hyp-(GalNAc- alkyl)3 dT 2-deoxythymidine-3′-phosphate dC 2′-deoxycytidine-3′-phosphate Y44 inverted abasic DNA (2-hydroxymethyl-tetrahydrofurane-5-phosphate) (Tgn) Thymidine-glycol nucleic acid (GNA) S-Isomer P Phosphate VP Vinyl-phosphate (Aam) 2′-O-(N-methylacetamide)adenosine-3′-phosphate

TABLE 3 Unmodified Sense and Antisense Strand Sequences of IGFALS dsRNAs Position SEQ Position SEQ Duplex Sense Sense  in NM ID Antisense Antisense  in NM ID name name Sequence 4970.2 NO name Sequence 004970.2 NO AD-62728 A-125826 ACAGA 196-216 28 A-125827 AAAGA 194-216 85 UGAGC CGCUG UCAGC AGCUC GUCUU AUCUG U UGU AD-62741 A-125832 AGCUC 203-223 29 A-125833 AACUG 201-223 86 AGCGU CAAAA CUUUU GACGC GCAGU UGAGC U UCA AD-68729 A-138233 CUGUG 318-337 30 A-138234 UUUGU 316-337 87 GCUGG C21A UGCCG C21A ACGGC UCCAG AACAA CCACA A GGG AD-68720 A-138215 GGACG 326-345 31 A-138216 UACGA 324-345 88 GCAAC C21A GAGGU C21A AACCU UGUUG CUCGU CCGUC A CAG AD-68717 A-138209 AACCG 549-568 32 A-138210 UUCCA 547-568 89 UCUGA G21A GCCUG G21A GCAGG CUCAG CUGGA ACGGU A UGU AD-62737 A-125830 GGACC 558-578 33 A-125831 UUCCA 556-578 90 UCAAC ACCCA CUGGG GGUUG UUGGA AGGUC A CCA AD-62713 A-125820 UGGCA 645-665 34 A-125821 AGGUA 643-665 91 ACAAA AGUCA CUGAC GUUUG UUACC UUGCC U AGC AD-62742 A-125786 GGUGC 678-698 35 A-125787 AGCCU 676-698 92 UGGCG GUUGC GGCAA CCGCC CAGGC AGCAC U CAG AD-62719 A-125838 GCUGG 747-767 36 A-125839 UCGUU 745-767 93 ACCUG C21A CCUGC C21A AGCAG UCAGG GAACG UCCAG A CUC AD-62724 A-125840 CUGGA 748-768 37 A-125841 UGCGU 746-768 94 CCUGA G21A UCCUG G21A GCAGG CUCAG AACGC GUCCA A GCU AD-68728 A-138231 GGCCA 776-795 38 A-138232 AACAC 774-795 95 UCAAG GUUUG GCAAA CCUUG CGUGU AUGGC U CCG AD-62717 A-125806 GCAAC 833-853 39 A-125807 UCACG 831-853 96 CUCAU G21A GCAGC G21A CGCUG GAUGA CCGUG GGUUG A CGG AD-62731 A-125796 GCUGC 844-864 40 A-125797 UGCGC 842-864 97 CGUGG C21A CCGGG C21A CCCCG GCCAC GGCGC GGCAG A CGA AD-62726 A-125794 UGGCC 851-871 41 A-125795 UCAGG 849-871 98 CCGGG G21A AAGGC G21A CGCCU GCCCG UCCUG GGGCC A ACG AD-68727 A-138229 GGGCC 872-891 42 A-138230 UAUCG 870-891 99 UGAAG G21A CAGCG G21A GCGCU CCUUC GCGAU AGGCC A CAG AD-62715 A-125774 GCGUG 911-931 43 A-125775 UCUCC 909-931 100 GCUGG G21A AGGAG G21A CCUCC GCCAG UGGAG CCACG A CGG AD-68710 A-138195 GGCCU 921-940 44 A-138196 UAACG 919-940 101 CCUGG C21A UGUCC C21A AGGAC UCCAG ACGUU GAGGC A CAG AD-62743 A-125802 UGGGC 1043-1063 45 A-125803 UCCGG 1041-1063 102 CACAA C21A AUGCG C21A CCGCA GUUGU UCCGG GGCCC A AGC AD-62711 A-125788 CCACA 1047-1067 46 A-125789 AGCUG 1045-1067 103 ACCGC CCGGA AUCCG UGCGG GCAGC UUGUG U GCC AD-68709 A-138193 AGCUG 1066-1085 47 A-138194 UAAAG 1064-1085 104 GCUGA G21A CUGCG G21A GCGCA CUCAG GCUUU CCAGC A UGC AD-68724 A-138223 CUCAC 1110-1129 48 A-138224 UUGGU 1108-1129 105 GCUAG G21A UGUGG G21A ACCAC UCUAG AACCA CGUGA A GCA AD-62734 A-125782 CCUCA 1161-1181 49 A-125783 AUGAC 1159-1181 106 CCAAC CGCCA GUGGC CGUUG GGUCA GUGAG U GCC AD-68111 A-135415 CCUCA 1161-1181 50 A-135416 AUGAC 1159-1181 107 CCAAC CGCCA GUGGC CGUUG GGUCA GUGAG U GCC AD-68719 A-138213 CACCA 1166-1185 51 A-138214 UUCAU 1164-1185 108 ACGUG GACCG GCGGU CCACG CAUGA UUGGU A GAG AD-68712 A-138199 ACCAA 1167-1186 52 A-138200 UUUCA 1165-1186 109 CGUGG C21A UGACC C21A CGGUC GCCAC AUGAA GUUGG A UGA AD-62730 A-125780 UCAUG 1178-1198 53 A-125781 AGUUC 1176-1198 110 AACCU CCAGA CUCUG GAGGU GGAAC UCAUG U ACC AD-68711 A-138197 ACCUC 1186-1205 54 A-138198 UGAGA 1184-1205 111 UCUGG C21A CAGUU C21A GAACU CCCAG GUCUC AGAGG A UUC AD-68713 A-138201 CUCUC 1188-1207 55 A-138202 UCGGA 1186-1207 112 UGGGA G21A GACAG G2 ACUGU UUCCC lA CUCCG AGAGA A GGU AD-62732 A-125812 GAACU 1194-1214 56 A-125813 UGAAG 1192-1214 113 GUCUC C21A GUUCC C21A CGGAA GGAGA CCUUC CAGUU A CCC AD-68715 A-138205 GGAAC 1195-1214 57 A-138206 UAAGG 1193-1214 114 UGUCU C21A UUCCG C21A CCGGA GAGAC ACCUU AGUUC A CCA AD-62738 A-125784 CUGUC 1197-1217 58 A-125785 UCCGG 1195-1217 115 UCCGG AAGGU AACCU UCCGG UCCGG AGACA A GUU AD-62736 A-125814 GUCUC 1199-1219 59 A-125815 UCUCC 1197-1219 116 CGGAA C21A GGAAG C2 CCUUC GUUCC lA CGGAG GGAGA A CAG AD-62712 A-125804 CGGAA 1204-1224 60 A-125805 UACCU 1202-1224 117 CCUUC G21A GCUCC G21A CGGAG GGAAG CAGGU GUUCC A GGA AD-68723 A-138221 AACCU 1209-1228 61 A-138222 UAACA 1207-1228 118 UCCGG C21A CCUGC C21A AGCAG UCCGG GUGUU AAGGU A UCC AD-62739 A-125800 CUUCC 1210-1230 62 A-125801 UCGGA 1208-1230 119 GGAGC G21A ACACC G21A AGGUG UGCUC UUCCG CGGAA A GGU AD-62723 A-125824 CAUCU 1302-1322 63 A-125825 UGUUC 1300-1322 120 CCAGC UUCGA AUCGA UGCUG AGAAC GAGAU A GCU AD-62745 A-125834 CUCCA 1305-1325 64 A-125835 UUCUG 1303-1325 121 GCAUC G21A UUCUU G21A GAAGA CGAUG ACAGA CUGGA A GAU AD-62733 A-125828 CAGCA 1308-1328 65 A-125829 AGGCU 1306-1328 122 UCGAA CUGUU GAACA CUUCG GAGCC AUGCU U GGA AD-68718 A-138211 UUCCU 1329-1348 66 A-138212 UAGGC 1327-1348 123 CAAGG C21A CGUUG C21A ACAAC UCCUU GGCCU GAGGA A AGA AD-62744 A-125818 AAGGA 1333-1353 67 A-125819 UCCCA 1331-1353 124 CAACG C21A CGAGG C2 GCCUC CCGUU lA GUGGG GUCCU A UGA AD-68721 A-138217 GCUGC 1388-1407 68 A-138218 UUCAG 1386-1407 125 UGGAG C21A GUCGA C21A CUCGA GCUCC CCUGA AGCAG A CUC AD-62727 A-125810 UGACC 1403-1423 69 A-125811 UCGUG 1401-1423 126 UCCAA C21A AGCUG C21A CCAGC GUUGG UCACG AGGUC A AGG AD-62740 A-125816 CAACC 1410-1430 70 A-125817 UGCAG 1408-1430 127 AGCUC C21A GUGCG C21A ACGCA UGAGC CCUGC UGGUU A GGA AD-62716 A-125790 UGGAG 1460-1480 71 A-125791 UGGAG 1458-1480 128 UACCU C21A AGCAG C21A GCUGC CAGGU UCUCC ACUCC A AGC AD-62725 A-125778 UGCAG 1523-1543 72 A-125779 UCAGC 1521-1543 129 CGGGC G21A CAGAA G21A CUUCU GGCCC GGCUG GCUGC A AGG AD-62714 A-125836 GCAGC 1524-1544 73 A-125837 UCCAG 1522-1544 130 GGGCC CCAGA UUCUG AGGCC GCUGG CGCUG A CAG AD-68716 A-138207 UUCUG 1536-1555 74 A-138208 UUGCG 1534-1555 131 GCUGG C21A AGACG C21A ACGUC UCCAG UCGCA CCAGA A AGG AD-62721 A-125792 GGCUG 1538-1558 75 A-125793 UGUUG 1536-1558 132 GACGU C21A UGCGA C21A CUCGC GACGU ACAAC CCAGC A CAG AD-62718 A-125822 UCAGG 1577-1597 76 A-125823 UCUGC 1575-1597 133 AAUAA AAGGA CUCCU GUUAU UGCAG UCCUG A AGG AD-68722 A-138219 UCAGC 1615-1634 77 A-138220 UUGAG 1613-1634 134 CUCAG C21A UUGUU C21A GAACA CCUGA ACUCA GGCUG A AGG AD-68725 A-138225 CAGCC 1616-1635 78 A-138226 AGUGA 1614-1635 135 UCAGG GUUGU AACAA UCCUG CUCAC AGGCU U GAG AD-68714 A-138203 AGCCU 1617-1636 79 A-138204 UAGUG 1615-1636 136 CAGGA G21A AGUUG G21A ACAAC UUCCU UCACU GAGGC A UGA AD-62722 A-125808 UCCAG 1766-1786 80 A-125809 UCCCC 1764-1786 137 GCCAU G21A UCACA G2 CUGUG GAUGG lA AGGGG CCUGG A ACG AD-62735 A-125798 GGGGA 1956-1976 81 A-125799 UGACA 1954-1976 138 CAGGU C21A CUGAG C21A CCUCA GACCU GUGUC GUCCC A CAG AD-68731 A-138237 UGUCA 2054-2073 82 A-138238 UUUUG 2052-2073 139 UCAAU G21A CCUUU G21A UAAAG AAUUG GCAAA AUGAC A AGC AD-68730 A-138235 UCAAU 2059-2078 83 A-138236 AUUGC 2057-2078 140 UAAAG CUUUG GCAAA CCUUU GGCAA AAUUG U AUG AD-68726 A-138227 AAAGG 2065-2084 84 A-138228 UAUUC 2063-2084 141 CAAAG C21A GAUUG C21A GCAAU CCUUU CGAAU GCCUU A UAA

TABLE 4 IGFALS Single Dose Screen in Hep3B Cells Hep3B 10 nM 10 nM 1 nM 1 nM 0.1 nM 0.1 nM DuplexID Avg SD Avg SD Avg SD AD-68729 53.9 16.0 57.5 3.8 97.4 7.0 AD-68720 48.4 4.1 73.2 27.9 116.3 9.3 AD-68717 22.6 5.6 55.4 8.3 101.8 26.9 AD-62742 126.5 20.7 ND 124.7 13.1 AD-62719 106.8 25.1 ND 66.5 4.7 AD-62724 87.8 8.4 ND 75.1 5.7 AD-68728 56.3 7.6 78.6 4.4 110.6 26.0 AD-62717 98.6 1.7 ND 102.9 56.2 AD-62731 105.7 3.4 ND 51.1 18.3 AD-62726 70.7 37.7 ND 93.5 8.9 AD-68727 68.9 14.6 94.8 12.8 124.7 23.3 AD-62715 118.5 41.8 ND 128.4 17.9 AD-68710 91.0 34.2 91.7 14.2 90.5 1.1 AD-62743 81.6 18.1 ND 123.9 20.9 AD-62711 107.0 11.3 ND 92.0 11.5 AD-68709 42.4 2.2 38.1 1.4 76.1 1.4 AD-68724 53.5 16.7 40.9 7.9 73.8 1.2 AD-62734 43.1 29.6 ND 115.2 0.3 AD-68111 29.5 14.8 37.5 0.7 92.1 3.9 AD-68719 45.4 18.9 59.6 5.7 108.7 25.0 AD-68712 40.8 1.9 58.0 4.7 97.4 13.1 AD-62730 98.4 14.2 ND 119.5 17.2 AD-68711 97.3 23.0 86.6 5.7 110.7 14.8 AD-68713 79.6 10.0 93.6 23.9 103.3 4.2 AD-62732 89.0 13.3 ND 66.0 14.3 AD-68715 48.7 13.1 71.9 6.8 78.7 7.2 AD-62738 133.2 34.5 ND 123.4 13.0 AD-62736 113.6 22.9 ND 84.9 13.9 AD-62712 68.5 22.6 ND 96.9 25.6 AD-68723 83.3 14.5 84.4 13.8 71.8 25.4 AD-62739 99.4 13.8 ND 105.6 24.4 AD-68718 83.1 6.8 78.2 0.1 119.8 36.8 AD-62744 98.8 10.4 ND 139.1 24.7 AD-68721 61.8 8.0 81.3 4.2 99.2 14.2 AD-62727 91.6 25.9 ND 86.6 46.5 AD-62740 138.9 16.0 ND 117.3 55.5 AD-62716 81.5 0.2 ND 127.8 20.9 AD-62725 109.1 6.1 ND 103.8 17.0 AD-62714 73.9 8.9 ND 64.0 16.0 AD-68716 18.2 0.3 28.6 17.6 40.2 10.8 AD-62721 62.0 7.7 ND 91.7 6.1 AD-68722 19.2 0.5 51.1 0.7 53.4 22.2 AD-68725 20.3 7.5 23.0 6.2 67.6 15.6 AD-68714 58.9 2.8 73.5 19.0 92.6 8.9 AD-62722 120.7 69.9 ND 115.0 25.4 AD-62735 60.2 29.8 ND 100.2 22.6 AD-68731 33.5 27.2 11.8 0.9 26.6 7.4 AD-68730 14.5 0.9 24.5 7.5 44.4 8.3 AD-68726 17.0 10.0 28.6 11.8 64.5 3.7 AD-62728 46.3 20.5 ND 49.5 10.2 AD-62741 116.8 48.6 ND 143.6 35.9 AD-62737 94.7 8.6 ND 75.2 55.1 AD-62713 83.0 20.3 ND 89.3 31.9 AD-62723 103.5 32.2 ND 66.5 22.2 AD-62745 66.7 4.4 ND 85.7 61.2 AD-62733 107.1 1.3 ND 35.9 3.8 AD-62718 129.6 42.7 ND 87.7 39.2 AD-1955 102.5 25.0 Mock 103.0 18.8 Naive 118.0 23.5 Data are expressed as percent message remaining relative to AD-1955 non-targeting control.

TABLE 5 IGFALS Modified Sequences Start Posi- tion Anti Anti in Oligo Sense SEQ sense sense  SEQ SEQ Duplex Sense  Se- ID Oligo Se- ID ID Name Name quence NO Name quence NO NO:1 AD- A- csusg 142 A- usUfs 199 316 68729 138233 uggCf 138234 uguUf uGfGf gCfCf Afcgg guccA caaca fgCfc aaL96 acags gsg AD- A- gsgsa 143 A- usAfs 200 324 68720 138215 cggCf 138216 cgaGf aAfCf aGfGf Afacc uuguU ucucg fgCfc uaL96 guccs asg AD- A- asasc 144 A- usUfs 201 547 68717 138209 cguCf 138210 ccaGf uGfAf cCfUf Gfcag gcucA gcugg fgAfc aaL96 gguus gsu AD- A- Gfsgs 145 A- asGfs 202 676 62742 125786 UfgCf 125787 cCfuG uGfgC fuUfg fGfGf Cfccg gCfaA CfcAf fcAfg gCfaC GfcUf fcsas L96 g AD- A- Gfscs 146 A- usCfs 203 745 62719 125838 UfgGf 125839 gUfuC aCfcU fcUfg fGfAf Cfuca gCfaG GfgUf fgAfa cCfaG CfgAf fcsus L96 c AD- A- Cfsus 147 A- usGfs 204 746 62724 125840 GfgAf 125841 cGfuU cCfuG fcCfu fAfGf Gfcuc cAfgG AfgGf faAfc uCfcA GfcAf fgscs L96 u AD- A- gsgsc 148 A- asAfs 205 774 68728 138231 cauCf 138232 cacGf aAfGf uUfUf Gfcaa gccuU acgug fgAfu uuL96 ggccs csg AD- A- Gfscs 149 A- usCfs 206 831 62717 125806 AfaCf 125807 aCfgG cUfcA fcAfg fUfCf Cfgau gCfuG GfaGf fcCfg gUfuG UfgAf fcsgs L96 g AD- A- Gfscs 150 A- usGfs 207 842 62731 125796 UfgCf 125797 cGfcC cGfuG fcGfg fGfCf Gfgcc cCfcG AfcGf fgGfc gCfaG GfcAf fcsgs L96 a AD- A- Ufsgs 151 A- usCfs 208 849 62726 125794 GfcCf 125795 aGfgA cCfgG faGfg fGfCf Cfgcc gCfcU CfgGf fuCfc gGfcC UfgAf fascs L96 g AD- A- gsgsg 152 A- usAfs 209 870 68727 138229 ccuGf 138230 ucgCf aAfGf aGfCf Gfcgc gccuU ugcga fcAfg uaL96 gcccs asg AD- A- Gfscs 153 A- usCfs 210 909 62715 125774 GfuGf 125775 uCfcA gCfuG fgGfa fGfCf Gfgcc cUfcC AfgCf fuGfg cAfcG AfgAf fcsgs L96 g AD- A- gsgsc 154 A- usAfs 211 919 68710 138195 cucCf 138196 acgUf uGfGf gUfCf Afgga cuccA cacgu fgGfa uaL96 ggccs asg AD- A- Ufsgs 155 A- usCfs 212 1041 62743 125802 GfgCf 125803 cGfgA cAfcA fuGfc fAfCf Gfguu cGfcA GfuGf fuCfc gCfcC GfgAf fasgs L96 c AD- A- Cfscs 156 A- asGfs 213 1045 62711 125788 AfcAf 125789 cUfgC aCfcG fcGfg fCfAf Afugc uCfcG GfgUf fgCfa uGfuG GfcUf fgscs L96 c AD- A- asgsc 157 A- usAfs 214 1064 68709 138193 uggCf 138194 aagCf uGfAf uGfCf Gfcgc gcucA agcuu fgCfc uaL96 agcus gsc AD- A- csusc 158 A- usUfs 215 1108 68724 138223 acgCf 138224 gguUf uAfGf gUfGf Afcca gucuA caacc fgCfg aaL96 ugags csa AD- A- Cfscs 159 A- asUfs 216 1159 62734 125782 UfcAf 125783 gAfcC cCfaA fgCfc fCfGf Afcgu uGfgC UfgGf fgGfu uGfaG CfaUf fgscs L96 c AD- A- cscsu 160 A- asUfs 217 1159 68111 135415 cacCf 135416 gacCf aAfCf gCfCf Gfugg acguU cgguc fgGfu auL96 gaggs csc AD- A- csasc 161 A- usUfs 218 1164 68719 138213 caaCf 138214 cauGf gUfGf aCfCf Gfcgg gccaC ucaug fgUfu aaL96 ggugs asg AD- A- ascsc 162 A- usUfs 219 1165 68712 138199 aacGf 138200 ucaUf uGfGf gAfCf Cfggu cgccA cauga fcGfu aaL96 uggus gsa AD- A- Ufscs 163 A- asGfs 220 1176 62730 125780 AfuGf 125781 uUfcC aAfcC fcAfg fUfCf Afgag uCfuG GfuUf fgGfa cAfuG AfcUf fascs L96 c AD- A- ascsc 164 A- usGfs 221 1184 68711 138197 ucuCf 138198 agaCf uGfGf aGfUf Gfaac ucccA ugucu fgAfg caL96 aggus usc AD- A- csusc 165 A- usCfs 222 1186 68713 138201 ucuGf 138202 ggaGf gGfAf aCfAf Afcug guucC ucucc fcAfg gaL96 agags gsu AD- A- Gfsas 166 A- usGfs 223 1192 62732 125812 AfcUf 125813 aAfgG gUfcU fuUfc fCfCf Cfgga gGfaA GfaCf fcCfu aGfuU UfcAf fcscs L96 c AD- A- gsgsa 167 A- usAfs 224 1193 68715 138205 acuGf 138206 aggli uCfUf fuCfC Cfcgg fggag aaccu AfcAf uaL96 guucc scsa AD- A- Cfsus 168 A- usCfs 225 1195 62738 125784 GfuCf 125785 cGfgA uCfcG faGfg fGfAf Ufucc aCfcU GfgAf fuCfc gAfcA GfgAf fgsus L96 u AD- A- Gfsus 169 A- usCfs 226 1197 62736 125814 CfuCf 125815 uCfcG cGfgA fgAfa fAfCf Gfguu cUfuC CfcGf fcGfg gAfgA AfgAf fcsas L96 g AD- A- Cfsgs 170 A- usAfs 227 1202 62712 125804 GfaAf 125805 cCfuG cCfuU fcUfc fCfCf Cfgga gGfaG AfgGf fcAfg uUfcC GfuAf fgsgs L96 a AD- A- asasc 171 A- usAfs 228 1207 68723 138221 cuuCf 138222 acaCf cGfGf cUfGf Afgca cuccG ggugu fgAfa uaL96 gguus csc AD- A- Cfsus 172 A- usCfs 229 1208 62739 125800 UfcCf 125801 gGfaA gGfaG fcAfc fCfAf Cfugc gGfuG UfcCf fuUfc gGfaA CfgAf fgsgs L96 u AD- A- ususc 173 A- usAfs 230 1327 68718 138211 cucAf 138212 ggcCf aGfGf gUfUf Afcaa guccU cggcc fuGfa uaL96 ggaas gsa AD- A- Afsas 174 A- usCfs 231 1331 62744 125818 GfgAf 125819 cCfaC cAfaC fgAfg fGfGf Gfccg cCfuC UfuGf fgUfg uCfcU GfgAf fusgs L96 a AD- A- gscsu 175 A- usUfs 232 1386 68721 138217 gcuGf 138218 cagGf gAfGf uCfGf Cfucg agcuC accug fcAfg aaL96 cagcs usc AD- A- Ufsgs 176 A- usCfs 233 1401 62727 125810 AfcCf 125811 gUfgA uCfcA fgCfu fAfCf Gfguu cAfgC GfgAf fuCfa gGfuC CfgAf fasgs L96 g AD- A- Cfsas 177 A- usGfs 234 1408 62740 125816 AfcCf 125817 cAfgG aGfcU fuGfc fCfAf Gfuga cGfcA GfcUf fcCfu gGfuU GfcAf fgsgs L96 a AD- A- Ufsgs 178 A- usGfs 235 1458 62716 125790 GfaGf 125791 gAfgA uAfcC fgCfa fUfGf Gfcag cUfgC GfuAf fuCfu cUfcC CfcAf fasgs L96 c AD- A- Ufsgs 179 A- usCfs 236 1521 62725 125778 CfaGf 125779 aGfcC cGfgG faGfa fCfCf Afggc uUfUf CfcGf gGfcU cUfgC fgAfL fasgs 96 g AD- A- Gfscs 180 A- usCfs 237 1522 62714 125836 AfgCf 125837 cAfgC gGfgC fcAfg fCfUf Afagg uCfuG CfcCf fgCfu gCfuG GfgAf fcsas L96 g AD- A- ususc 181 A- usUfs 238 1534 68716 138207 uggCf 138208 gcgAf uGfGf gAfCf Afcgu guccA cucgc fgCfc aaL96 agaas gsg AD- A- Gfsgs 182 A- usGfs 239 1536 62721 125792 CfuGf 125793 uUfgU gAfcG fgCfg fUfCf Afgac uCfgC GfuCf faCfa cAfgC AfcAf fcsas L96 g AD- A- uscsa 183 A- usUfs 240 1613 68722 138219 gccUf 138220 gagUf cAfGf uGfUf Gfaac uccuG aacuc faGfg aaL96 cugas gsg AD- A- csasg 184 A- asGfs 241 1614 68725 138225 ccuCf 138226 ugaGf aGfGf uUfGf Afaca uuccU acuca fgAfg cuL96 gcugs asg AD- A- asgsc 185 A- usAfs 242 1615 68714 138203 cucAf 138204 gugAf gGfAf gUfUf Afcaa guucC cucac fuGfa uaL96 ggcus gsa AD- A- Ufscs 186 A- usCfs 243 1764 62722 125808 CfaGf 125809 cCfcU gCfcA lcAfc fUfCf Afgau uGfuG GfgCf faGfg cUfgG GfgAf fascs L96 g AD- A- Gfsgs 187 A- usGfs 244 1954 62735 125798 GfgAf 125799 aCfaC cAfgG fuGfa fUfCf Gfgac cUfcA CfuGf fgUfg uCfcC UfcAf fcsas L96 g AD- A- usgsu 188 A- usUfs 245 2052 68731 138237 cauCf 138238 uugCf aAfUf cUfUf Ufaaa uaauU ggcaa fgAfu aaL96 gacas gsc AD- A- uscsa 189 A- asUfs 246 2057 68730 138235 auuAf 138236 ugcCf aAfGf uUfUf Gfcaa gccuU aggca fuAfa auL96 uugas usg AD- A- asasa 190 A- usAfs 247 2063 68726 138227 ggcAf 138228 uucGf aAfGf aUfUf Gfcaa gccuU ucgaa fuGfc uaL96 cuuus asa AD- A- Afscs 191 A- asAfs 248 774 62728 125826 AfgAf 125827 aGfaC uGfaG fgCfu fCfUf Gfagc cAfgC UfcAf fgUfc uCfuG UfuUf fusgs L96 u AD- A- Afsgs 192 A- asAfs 249 201 62741 125832 CfuCf 125833 cUfgC aGfcG faAfa fUfCf Afgac uUfuU GfcUf fgCfa gAfgC GfuUf fuscs L96 a AD- A- Gfsgs 193 A- usUfs 250 556 62737 125830 AfcCf 125831 cCfaA uCfaA fcCfc fCfCf Afggu uGfgG UfgAf fuUfg gGfuC GfaAf fcscs L96 a AD- A- Ufsgs 194 A- asGfs 251 643 62713 125820 GfcAf 125821 gUfaA aCfaA fgUfc fAfCf Afguu uGfaC UfgUf fuUfa uGfcC CfcUf fasgs L96 c AD- A- Cfsas 195 A- usGfs 252 1300 62723 125824 UfcUf 125825 uUfcl cCfaG ifuCf fCfAf gAfug uCfgA cUfgG faGfa faGfa AfcAf Ufgsc L96 su AD- A- Cfsus 196 A- usUfs 253 1301 62745 125834 CfcAf 125835 cUfgU gCfaU fuCfu fCfGf Ufcga aAfgA UfgCf faCfa uGfgA GfaAf fgsas L96 u AD- A- Cfsas 197 A- asGfs 254 1306 62733 125828 GfcAf 125829 gCfuC uCfgA fuGfu fAfGf Ufcuu aAfcA CfgAf fgAfg uGfcU CfcUf fgsgs L96 a AD- A- Ufscs 198 A- usCfs 255 1575 62718 125822 AfgGf 125823 uGfcA aAfuA faGfg fAfCf Afguu uCfcU AfuUf fuGf cCfuG cAfgA fasgs fL96 g

Example 3—Knockdown of IGFALs Expression with an IGFALS siRNA Decreases Expression of IGF-1

A series of siRNAs targeting mouse IGFALS were designed and tested for the ability to knockdown expression of IGFALs mRNA in 6-8 week old C57Bl/6 female mice (n=3 per group). A single 10 mg/kg dose of AD-62713, AD-62724, AD-62745, or AD-62728; or PBS control, was administered subcutaneously on day 1. On day 7, the mice were sacrificed to assess knockdown of IGFLALS mRNA in liver and IGFALS and IGF-1 protein in serum.

AD-62728 was found to be most effective in decreasing expression of IGFALS mRNA and protein. Specifically, at day 7, IFGALS mRNA expression in the liver was found to be about 15% of the PBS control. At day 7 after treatment with AD-62713 and AD-62745, IGFALS mRNA expression in the liver was found to be about 65% of the PBS control for both duplexes.

A decrease in serum IGFALS protein levels was found to correspond to the decrease in IGFALS mRNA in the liver. Specifically, AD-62728 decreased the serum IGFALS protein level to about 3.9 μg/ml, as compared to about 6.4 μg/ml in the PBS control. AD-62713 and AD-62745 decreased the serum IGFALS level to about 5.2 μg/ml and 4.6 μg/ml, respectively.

A decrease in serum IGF-1 was also observed in response to treatment with the duplexes. Specifically, AD-62727 decreased the serum IGF-1 protein level to about 13 ng/ml as compared to about 34 ng/ml in the PBS control. AD-62713 and AD-62745 decreased serum IGF-1 levels to about 20 ng/ml and 27 ng/ml, respectively.

Further, in a multidose study, AD-62728 was demonstrated to be effective in knockdown of expression of IGFALS mRNA in liver in an expected dose response manner. Specifically, C57Bl/6 female mice, 6-8 weeks of age (n=3 per group) were administered either four doses of AD-62728 at 1 mg/kg or 3 mg/kg once weekly, or two doses at 3 mg/kg or 10 mg/kg every other week; or a PBS control. IGFALS mRNA knockdown was observed in the expected dose response manner.

Example 4—In Vitro Screening Bioinformatics

A set of double stranded RNAi agents targeting human IGFALS (human NCBI refseq ID: NM_004970; NCBI GeneID: 3483, SEQ ID NO: 1) were designed using custom R and Python scripts. The human IGFALS REFSEQ mRNA has a length of 2168 bases.

The rationale and method for the set of agent designs is as follows: the predicted efficacy for every potential 19mer siRNA from position 10 through position 2168 was determined with a linear model derived the direct measure of mRNA knockdown from more than 20,000 distinct siRNA designs targeting a large number of vertebrate genes. The custom Python script built the set of agents by systematically selecting a siRNA every 11 bases along the target mRNA starting at position 10. At each of the positions, the neighboring agent (one position to the 5′ end of the mRNA, one position to the 3′ end of the mRNA) was swapped into the design set if the predicted efficacy was better than the efficacy at the exact every-11th siRNA. Low complexity agents, i.e., those with Shannon Entropy measures below 1.35 were excluded from the set.

In Vitro Dual-Glo® Screening Cell Culture and Transfections

Cos7 cells (ATCC, Manassas, Va.) were grown to near confluence at 37° C. in an atmosphere of 5% CO₂ in DMEM (ATCC) supplemented with 10% FBS, before being released from the plate by trypsinization. Dual-Glo® Luciferase constructs were generated in the psiCHECK2 plasmid and contained approximately 2.0 kb (human) IGFALS sequences (SEQ ID NO: 23). Dual-luciferase plasmids were co-transfected with double stranded agents into 3000 cells using Lipofectamine RNAiMax (Invitrogen, Carlsbad Calif. cat #13778-150). For each well of a 384 well plate, 0.1 μl of Lipofectamine was added to 3 ng of plasmid vector and agent in 15 μl of Opti-MEM and allowed to complex at room temperature for 15 minutes. The mixture was then added to the cells resuspended in 35 ul of fresh complete media. Cells were incubated for 48 hours before luciferase was measured. Single dose experiments were performed at 10 nM final duplex concentration.

Dual-Glo® Luciferase Assay

Forty-eight hours after the siRNAs were transfected, Firefly (transfection control) and Renilla (fused to IGFALS target sequence in 3′ UTR, SEQ ID NO: 23) luciferase were measured. First, media was removed from cells. Then Firefly luciferase activity was measured by adding 20 μl of Dual-Glo® Luciferase Reagent mixed with 20 μl of complete media to each well. The mixture was incubated at room temperature for 30 minutes before luminescense (500 nm) was measured on a Spectramax (Molecular Devices) to detect the Firefly luciferase signal. Renilla luciferase activity was measured by adding 20 ul of room temperature of Dual-Glo® Stop & Glo® Reagent to each well and the plates were incubated for 20 minutes before luminescence was again measured to determine the Renilla luciferase signal. The Dual-Glo® Stop & Glo® Reagent quenched the firefly luciferase signal and sustained luminescence for the Renilla luciferase reaction. Double stranded RNAi agent activity was determined by normalizing the Renilla (IGFALS) signal to the Firefly (control) signal within each well. The magnitude of agent activity was then assessed relative to cells that were transfected with the same vector but were not treated with agent or were treated with a non-targeting double stranded RNAi agent. All transfections were done in quadruplicates.

TABLE 6 Unmodified Sense and Antisense Strand Sequences of IGFALS dsRNAs  Sense  SEQ Antisense SEQ  Range in Duplex Oligo Sense oligo  ID Oligo Antisense oligo ID SEQ ID Name Name sequence NO Range Name sequence NO NO: 1 AD-73764 A-147667 AGGGCAGGGGUGGCCGGCA 256 11-29 A-147668 UGCCGGCCACCCCUGCCCU 441 11-29 AD-73765 A-147669 CCGGCACAGCAGACGUACA 257 24-42 A-147670 UGUACGUCUGCUGUGCCGG 442 24-42 AD-73766 A-147671 AGACGUACCCUCCCUCGCU 258 34-52 A-147672 AGCGAGGGAGGGUACGUCU 443 34-52 AD-73767 A-147673 UCCCUCGCUGCCUGCCUGA 259 44-62 A-147674 UCAGGCAGGCAGCGAGGGA 444 44-62 AD-73768 A-147675 UGCCUGCAGCCUGCCCUGA 260 56-74 A-147676 UCAGGGCAGGCUGCAGGCA 445 56-74 AD-73769 A-147677 UGCCCUGCAUGCAGGAUGA 261 67-85 A-147678 UCAUCCUGCAUGCAGGGCA 446 67-85 AD-73770 A-147679 AGGAUGGCCCUGAGGAAAG 262 79-97 A-147680 CUUUCCUCAGGGCCAUCCU 447 79-97 AD-73771 A-147681 CUGAGGAAAGGAGGCCUGA 263  88-106 A-147682 UCAGGCCUCCUUUCCUCAG 448  88-106 AD-73772 A-147683 AGGCCUGGCCCUGGCGCUA 264  99-117 A-147684 UAGCGCCAGGGCCAGGCCU 449  99-117 AD-73773 A-147685 GCGCUGCUGCUGCUGUCCU 265 112-130 A-147686 AGGACAGCAGCAGCAGCGC 450 112-130 AD-73774 A-147687 UGCUGUCCUGGGUGGCACU 266 122-140 A-147688 AGUGCCACCCAGGACAGCA 451 122-140 AD-73775 A-147689 UGGCACUGGGCCCCCGCAA 267 134-152 A-147690 UUGCGGGGGCCCAGUGCCA 452 134-152 AD-73776 A-147691 GCCCCCGCAGCCUGGAGGA 268 143-161 A-147692 UCCUCCAGGCUGCGGGGGC 453 143-161 AD-73777 A-147693 UGGAGGGAGCAGACCCCGA 269 155-173 A-147694 UCGGGGUCUGCUCCCUCCA 454 155-173 AD-73778 A-147695 AGACCCCGGAACGCCGGGA 270 165-183 A-147696 UCCCGGCGUUCCGGGGUCU 455 165-183 AD-73779 A-147697 CGCCGGGGGAAGCCGAGGA 271 176-194 A-147698 UCCUCGGCUUCCCCCGGCG 456 176-194 AD-73780 A-147699 CGAGGGCCCAGCGUGCCCA 272 189-207 A-147700 UGGGCACGCUGGGCCCUCG 457 189-207 AD-73781 A-147701 AGCGUGCCCGGCCGCCUGU 273 198-216 A-147702 ACAGGCGGCCGGGCACGCU 458 198-216 AD-73782 A-147703 GCCUGUGUCUGCAGCUACA 274 211-229 A-147704 UGUAGCUGCAGACACAGGC 459 211-229 AD-73783 A-147705 UGCAGCUACGAUGACGACA 275 220-238 A-147706 UGUCGUCAUCGUAGCUGCA 460 220-238 AD-73784 A-147707 GACGACGCGGAUGAGCUCA 276 232-250 A-147708 UGAGCUCAUCCGCGUCGUC 461 232-250 AD-73785 A-147709 AUGAGCUCAGCGUCUUCUA 277 242-260 A-147710 UAGAAGACGCUGAGCUCAU 462 242-260 AD-73786 A-147711 UCUUCUGCAGCUCCAGGAA 278 254-272 A-147712 UUCCUGGAGCUGCAGAAGA 463 254-272 AD-73787 A-147713 UCCAGGAACCUCACGCGCA 279 265-283 A-147714 UGCGCGUGAGGUUCCUGGA 464 265-283 AD-73788 A-147715 UCACGCGCCUGCCUGAUGA 280 275-293 A-147716 UCAUCAGGCAGGCGCGUGA 465 275-293 AD-73789 A-147717 UGAUGGAGUCCCGGGCGGA 281 288-306 A-147718 UCCGCCCGGGACUCCAUCA 466 288-306 AD-73790 A-147719 CGGGCGGCACCCAAGCCCU 282 299-317 A-147720 AGGGCUUGGGUGCCGCCCG 467 299-317 AD-73791 A-147721 CAAGCCCUGUGGCUGGACA 283 310-328 A-147722 UGUCCAGCCACAGGGCUUG 468 310-328 AD-73792 A-147723 UGGCUGGACGGCAACAACA 284 319-337 A-147724 UGUUGUUGCCGUCCAGCCA 469 319-337 AD-73793 A-147725 AACAACCUCUCGUCCGUCA 285 331-349 A-147726 UGACGGACGAGAGGUUGUU 470 331-349 AD-73794 A-147727 UCCGUCCCCCCGGCAGCCU 286 343-361 A-147728 AGGCUGCCGGGGGGACGGA 471 343-361 AD-73795 A-147729 CGGCAGCCUUCCAGAACCU 287 353-371 A-147730 AGGUUCUGGAAGGCUGCCG 472 353-371 AD-73796 A-147731 CAGAACCUCUCCAGCCUGA 288 364-382 A-147732 UCAGGCUGGAGAGGUUCUG 473 364-382 AD-73797 A-147733 AGCCUGGGCUUCCUCAACA 289 376-394 A-147734 UGUUGAGGAAGCCCAGGCU 474 376-394 AD-73798 A-147735 UUCCUCAACCUGCAGGGCA 290 385-403 A-147736 UGCCCUGCAGGUUGAGGAA 475 385-403 AD-73799 A-147737 CAGGGCGGCCAGCUGGGCA 291 397-415 A-147738 UGCCCAGCUGGCCGCCCUG 476 397-415 AD-73800 A-147739 AGCUGGGCAGCCUGGAGCA 292 407-425 A-147740 UGCUCCAGGCUGCCCAGCU 477 407-425 AD-73801 A-147741 CUGGAGCCACAGGCGCUGA 293 418-436 A-147742 UCAGCGCCUGUGGCUCCAG 478 418-436 AD-73802 A-147743 CGCUGCUGGGCCUAGAGAA 294 431-449 A-147744 UUCUCUAGGCCCAGCAGCG 479 431-449 AD-73803 A-147745 CUAGAGAACCUGUGCCACA 295 442-460 A-147746 UGUGGCACAGGUUCUCUAG 480 442-460 AD-73804 A-147747 UGUGCCACCUGCACCUGGA 296 452-470 A-147748 UCCAGGUGCAGGUGGCACA 481 452-470 AD-73805 A-147749 ACCUGGAGCGGAACCAGCU 297 464-482 A-147750 AGCUGGUUCCGCUCCAGGU 482 464-482 AD-73806 A-147751 AACCAGCUGCGCAGCCUGA 298 475-493 A-147752 UCAGGCUGCGCAGCUGGUU 483 475-493 AD-73807 A-147753 CGCAGCCUGGCACUCGGCA 299 484-502 A-147754 UGCCGAGUGCCAGGCUGCG 484 484-502 AD-73808 A-147755 UCGGCACGUUUGCACACAA 300 497-515 A-147756 UUGUGUGCAAACGUGCCGA 485 497-515 AD-73809 A-147757 UUGCACACACGCCCGCGCU 301 506-524 A-147758 AGCGCGGGCGUGUGUGCAA 486 506-524 AD-73810 A-147759 CCCGCGCUGGCCUCGCUCA 302 517-535 A-147760 UGAGCGAGGCCAGCGCGGG 487 517-535 AD-73811 A-147761 UCGCUCGGCCUCAGCAACA 303 529-547 A-147762 UGUUGCUGAGGCCGAGCGA 488 529-547 AD-73812 A-147763 AGCAACAACCGUCUGAGCA 304 541-559 A-147764 UGCUCAGACGGUUGUUGCU 489 541-559 AD-73813 A-147767 CUGGAGGACGGGCUCUUCA 305 562-580 A-147768 UGAAGAGCCCGUCCUCCAG 490 562-580 AD-73814 A-147769 CUCUUCGAGGGCCUCGGCA 306 574-592 A-147770 UGCCGAGGCCCUCGAAGAG 491 574-592 AD-73815 A-147771 GGCCUCGGCAGCCUCUGGA 307 583-601 A-147772 UCCAGAGGCUGCCGAGGCC 492 583-601 AD-73816 A-147773 UCUGGGACCUCAACCUCGA 308 596-614 A-147774 UCGAGGUUGAGGUCCCAGA 493 596-614 AD-73817 A-147775 AACCUCGGCUGGAAUAGCA 309 607-625 A-147776 UGCUAUUCCAGCCGAGGUU 494 607-625 AD-73818 A-147777 UGGAAUAGCCUGGCGGUGA 310 616-634 A-147778 UCACCGCCAGGCUAUUCCA 495 616-634 AD-73819 A-147779 CGGUGCUCCCCGAUGCGGA 311 629-647 A-147780 UCCGCAUCGGGGAGCACCG 496 629-647 AD-73820 A-147781 GAUGCGGCGUUCCGCGGCA 312 640-658 A-147782 UGCCGCGGAACGCCGCAUC 497 640-658 AD-73821 A-147783 UUCCGCGGCCUGGGCAGCA 313 649-667 A-147784 UGCUGCCCAGGCCGCGGAA 498 649-667 AD-73822 A-147785 GCAGCCUGCGCGAGCUGGU 314 662-680 A-147786 ACCAGCUCGCGCAGGCUGC 499 662-680 AD-73823 A-147787 GAGCUGGUGCUGGCGGGCA 315 673-691 A-147788 UGCCCGCCAGCACCAGCUC 500 673-691 AD-73824 A-147789 CUGGCGGGCAACAGGCUGA 316 682-700 A-147790 UCAGCCUGUUGCCCGCCAG 501 682-700 AD-73825 A-147791 AGGCUGGCCUACCUGCAGA 317 694-712 A-147792 UCUGCAGGUAGGCCAGCCU 502 694-712 AD-73826 A-147793 ACCUGCAGCCCGCGCUCUU 318 704-722 A-147794 AAGAGCGCGGGCUGCAGGU 503 704-722 AD-73827 A-147795 CGCUCUUCAGCGGCCUGGA 319 716-734 A-147796 UCCAGGCCGCUGAAGAGCG 504 716-734 AD-73828 A-147797 CGGCCUGGCCGAGCUCCGA 320 726-744 A-147798 UCGGAGCUCGGCCAGGCCG 505 726-744 AD-73829 A-147799 AGCUCCGGGAGCUGGACCU 321 737-755 A-147800 AGGUCCAGCUCCCGGAGCU 506 737-755 AD-73830 A-147801 CUGGACCUGAGCAGGAACA 322 748-766 A-147802 UGUUCCUGCUCAGGUCCAG 507 748-766 AD-73831 A-147803 AGGAACGCGCUGCGGGCCA 323 760-778 A-147804 UGGCCCGCAGCGCGUUCCU 508 760-778 AD-73832 A-147805 CGGGCCAUCAAGGCAAACA 324 772-790 A-147806 UGUUUGCCUUGAUGGCCCG 509 772-790 AD-73833 A-147807 AAGGCAAACGUGUUCGUGA 325 781-799 A-147808 UCACGAACACGUUUGCCUU 510 781-799 AD-73834 A-147809 UUCGUGCAGCUGCCCCGGA 326 793-811 A-147810 UCCGGGGCAGCUGCACGAA 511 793-811 AD-73835 A-147813 AGAAACUCUACCUGGACCA 327 815-833 A-147814 UGGUCCAGGUAGAGUUUCU 512 815-833 AD-73836 A-147815 CCUGGACCGCAACCUCAUA 328 825-843 A-147816 UAUGAGGUUGCGGUCCAGG 513 825-843 AD-73837 A-147817 CCUCAUCGCUGCCGUGGCA 329 837-855 A-147818 UGCCACGGCAGCGAUGAGG 514 837-855 AD-73838 A-147819 CGUGGCCCCGGGCGCCUUA 330 849-867 A-147820 UAAGGCGCCCGGGGCCACG 515 849-867 AD-73839 A-147821 GGCGCCUUCCUGGGCCUGA 331 859-877 A-147822 UCAGGCCCAGGAAGGCGCC 516 859-877 AD-73840 A-147823 UGGGCCUGAAGGCGCUGCA 332 869-887 A-147824 UGCAGCGCCUUCAGGCCCA 517 869-887 AD-73841 A-147825 CGCUGCGAUGGCUGGACCU 333 881-899 A-147826 AGGUCCAGCCAUCGCAGCG 518 881-899 AD-73842 A-147827 UGGACCUGUCCCACAACCA 334 893-911 A-147828 UGGUUGUGGGACAGGUCCA 519 893-911 AD-73843 A-147829 CACAACCGCGUGGCUGGCA 335 904-922 A-147830 UGCCAGCCACGCGGUUGUG 520 904-922 AD-73844 A-147831 UGGCUGGCCUCCUGGAGGA 336 914-932 A-147832 UCCUCCAGGAGGCCAGCCA 521 914-932 AD-73845 A-147833 CCUGGAGGACACGUUCCCA 337 924-942 A-147834 UGGGAACGUGUCCUCCAGG 522 924-942 AD-73846 A-147835 UUCCCCGGUCUGCUGGGCA 338 937-955 A-147836 UGCCCAGCAGACCGGGGAA 523 937-955 AD-73847 A-147837 UGCUGGGCCUGCGUGUGCU 339 947-965 A-147838 AGCACACGCAGGCCCAGCA 524 947-965 AD-73848 A-147839 CGUGUGCUGCGGCUGUCCA 340 958-976 A-147840 UGGACAGCCGCAGCACACG 525 958-976 AD-73849 A-147841 CUGUCCCACAACGCCAUCA 341 970-988 A-147842 UGAUGGCGUUGUGGGACAG 526 970-988 AD-73850 A-147843 AACGCCAUCGCCAGCCUGA 342 979-997 A-147844 UCAGGCUGGCGAUGGCGUU 527 979-997 AD-73851 A-147845 AGCCUGCGGCCCCGCACCU 343  991-1009 A-147846 AGGUGCGGGGCCGCAGGCU 528  991-1009 AD-73852 A-147847 CGCACCUUCAAGGACCUGA 344 1003-1021 A-147848 UCAGGUCCUUGAAGGUGCG 529 1003-1021 AD-73853 A-147849 AAGGACCUGCACUUCCUGA 345 1012-1030 A-147850 UCAGGAAGUGCAGGUCCUU 530 1012-1030 AD-73854 A-147851 UUCCUGGAGGAGCUGCAGA 346 1024-1042 A-147852 UCUGCAGCUCCUCCAGGAA 531 1024-1042 AD-73855 A-147853 CUGCAGCUGGGCCACAACA 347 1036-1054 A-147854 UGUUGUGGCCCAGCUGCAG 532 1036-1054 AD-73856 A-147855 CCACAACCGCAUCCGGCAA 348 1047-1065 A-147856 UUGCCGGAUGCGGUUGUGG 533 1047-1065 AD-73857 A-147857 UCCGGCAGCUGGCUGAGCA 349 1058-1076 A-147858 UGCUCAGCCAGCUGCCGGA 534 1058-1076 AD-73858 A-147859 UGGCUGAGCGCAGCUUUGA 350 1067-1085 A-147860 UCAAAGCUGCGCUCAGCCA 535 1067-1085 AD-73859 A-147861 AGCUUUGAGGGCCUGGGGA 351 1078-1096 A-147862 UCCCCAGGCCCUCAAAGCU 536 1078-1096 AD-73860 A-147863 UGGGGCAGCUUGAGGUGCU 352 1091-1109 A-147864 AGCACCUCAAGCUGCCCCA 537 1091-1109 AD-73861 A-147865 UUGAGGUGCUCACGCUAGA 353 1100-1118 A-147866 UCUAGCGUGAGCACCUCAA 538 1100-1118 AD-73862 A-147867 ACGCUAGACCACAACCAGA 354 1111-1129 A-147868 UCUGGUUGUGGUCUAGCGU 539 1111-1129 AD-73863 A-147869 AACCAGCUCCAGGAGGUCA 355 1123-1141 A-147870 UGACCUCCUGGAGCUGGUU 540 1123-1141 AD-73864 A-147871 AGGAGGUCAAGGCGGGCGA 356 1133-1151 A-147872 UCGCCCGCCUUGACCUCCU 541 1133-1151 AD-73865 A-147873 CGGGCGCUUUCCUCGGCCU 357 1145-1163 A-147874 AGGCCGAGGAAAGCGCCCG 542 1145-1163 AD-73866 A-147875 CUCGGCCUCACCAACGUGA 358 1156-1174 A-147876 UCACGUUGGUGAGGCCGAG 543 1156-1174 AD-73867 A-147877 AACGUGGCGGUCAUGAACA 359 1168-1186 A-147878 UGUUCAUGACCGCCACGUU 544 1168-1186 AD-73868 A-147879 UCAUGAACCUCUCUGGGAA 360 1178-1196 A-147880 UUCCCAGAGAGGUUCAUGA 545 1178-1196 AD-73869 A-147881 UCUGGGAACUGUCUCCGGA 361 1189-1207 A-147882 UCCGGAGACAGUUCCCAGA 546 1189-1207 AD-73870 A-147883 UCUCCGGAACCUUCCGGAA 362 1200-1218 A-147884 UUCCGGAAGGUUCCGGAGA 547 1200-1218 AD-73871 A-147885 UUCCGGAGCAGGUGUUCCA 363 1211-1229 A-147886 UGGAACACCUGCUCCGGAA 548 1211-1229 AD-73872 A-147887 GGUGUUCCGGGGCCUGGGA 364 1221-1239 A-147888 UCCCAGGCCCCGGAACACC 549 1221-1239 AD-73873 A-147889 CUGGGCAAGCUGCACAGCA 365 1234-1252 A-147890 UGCUGUGCAGCUUGCCCAG 550 1234-1252 AD-73874 A-147891 UGCACAGCCUGCACCUGGA 366 1244-1262 A-147892 UCCAGGUGCAGGCUGUGCA 551 1244-1262 AD-73875 A-147895 CAGCUGCCUGGGACGCAUA 367 1266-1284 A-147896 UAUGCGUCCCAGGCAGCUG 552 1266-1284 AD-73876 A-147897 GACGCAUCCGCCCGCACAA 368 1277-1295 A-147898 UUGUGCGGGCGGAUGCGUC 553 1277-1295 AD-73877 A-147899 CGCACACCUUCACCGGCCU 369 1289-1307 A-147900 AGGCCGGUGAAGGUGUGCG 554 1289-1307 AD-73878 A-147901 UCACCGGCCUCUCGGGGCU 370 1298-1316 A-147902 AGCCCCGAGAGGCCGGUGA 555 1298-1316 AD-73879 A-147903 UCGGGGCUCCGCCGACUCU 371 1309-1327 A-147904 AGAGUCGGCGGAGCCCCGA 556 1309-1327 AD-73880 A-147905 CGACUCUUCCUCAAGGACA 372 1321-1339 A-147906 UGUCCUUGAGGAAGAGUCG 557 1321-1339 AD-73881 A-147907 CAAGGACAACGGCCUCGUA 373 1332-1350 A-147908 UACGAGGCCGUUGUCCUUG 558 1332-1350 AD-73882 A-147909 GGCCUCGUGGGCAUUGAGA 374 1342-1360 A-147910 UCUCAAUGCCCACGAGGCC 559 1342-1360 AD-73883 A-147911 UUGAGGAGCAGAGCCUGUA 375 1355-1373 A-147912 UACAGGCUCUGCUCCUCAA 560 1355-1373 AD-73884 A-147913 AGAGCCUGUGGGGGCUGGA 376 1364-1382 A-147914 UCCAGCCCCCACAGGCUCU 561 1364-1382 AD-73885 A-147915 GGGCUGGCGGAGCUGCUGA 377 1375-1393 A-147916 UCAGCAGCUCCGCCAGCCC 562 1375-1393 AD-73886 A-147917 UGCUGGAGCUCGACCUGAA 378 1388-1406 A-147918 UUCAGGUCGAGCUCCAGCA 563 1388-1406 AD-73887 A-147919 GACCUGACCUCCAACCAGA 379 1399-1417 A-147920 UCUGGUUGGAGGUCAGGUC 564 1399-1417 AD-73888 A-147921 UCCAACCAGCUCACGCACA 380 1408-1426 A-147922 UGUGCGUGAGCUGGUUGGA 565 1408-1426 AD-73889 A-147923 ACGCACCUGCCCCACCGCA 381 1420-1438 A-147924 UGCGGUGGGGCAGGUGCGU 566 1420-1438 AD-73890 A-147925 CACCGCCUCUUCCAGGGCA 382 1432-1450 A-147926 UGCCCUGGAAGAGGCGGUG 567 1432-1450 AD-73891 A-147927 UCCAGGGCCUGGGCAAGCU 383 1442-1460 A-147928 AGCUUGCCCAGGCCCUGGA 568 1442-1460 AD-73892 A-147929 GCAAGCUGGAGUACCUGCU 384 1454-1472 A-147930 AGCAGGUACUCCAGCUUGC 569 1454-1472 AD-73893 A-147931 UACCUGCUGCUCUCCCGCA 385 1465-1483 A-147932 UGCGGGAGAGCAGCAGGUA 570 1465-1483 AD-73894 A-147933 CUCUCCCGCAACCGCCUGA 386 1474-1492 A-147934 UCAGGCGGUUGCGGGAGAG 571 1474-1492 AD-73895 A-147935 CCGCCUGGCAGAGCUGCCA 387 1485-1503 A-147936 UGGCAGCUCUGCCAGGCGG 572 1485-1503 AD-73896 A-147937 AGCUGCCGGCGGACGCCCU 388 1496-1514 A-147938 AGGGCGUCCGCCGGCAGCU 573 1496-1514 AD-73897 A-147939 GACGCCCUGGGCCCCCUGA 389 1507-1525 A-147940 UCAGGGGGCCCAGGGCGUC 574 1507-1525 AD-73898 A-147941 CCCCUGCAGCGGGCCUUCU 390 1519-1537 A-147942 AGAAGGCCCGCUGCAGGGG 575 1519-1537 AD-73899 A-147943 GGGCCUUCUGGCUGGACGU 391 1529-1547 A-147944 ACGUCCAGCCAGAAGGCCC 576 1529-1547 AD-73900 A-147945 UGGACGUCUCGCACAACCA 392 1541-1559 A-147946 UGGUUGUGCGAGACGUCCA 577 1541-1559 AD-73901 A-147947 ACAACCGCCUGGAGGCAUU 393 1553-1571 A-147948 AAUGCCUCCAGGCGGUUGU 578 1553-1571 AD-73902 A-147949 GAGGCAUUGCCCAACAGCA 394 1564-1582 A-147950 UGCUGUUGGGCAAUGCCUC 579 1564-1582 AD-73903 A-147951 CAACAGCCUCUUGGCACCA 395 1575-1593 A-147952 UGGUGCCAAGAGGCUGUUG 580 1575-1593 AD-73904 A-147953 UUGGCACCACUGGGGCGGA 396 1585-1603 A-147954 UCCGCCCCAGUGGUGCCAA 581 1585-1603 AD-73905 A-147955 UGGGGCGGCUGCGCUACCU 397 1595-1613 A-147956 AGGUAGCGCAGCCGCCCCA 582 1595-1613 AD-73906 A-147957 CGCUACCUCAGCCUCAGGA 398 1606-1624 A-147958 UCCUGAGGCUGAGGUAGCG 583 1606-1624 AD-73907 A-147959 UCAGGAACAACUCACUGCA 399 1619-1637 A-147960 UGCAGUGAGUUGUUCCUGA 584 1619-1637 AD-73908 A-147961 CUCACUGCGGACCUUCACA 400 1629-1647 A-147962 UGUGAAGGUCCGCAGUGAG 585 1629-1647 AD-73909 A-147963 ACCUUCACGCCGCAGCCCA 401 1639-1657 A-147964 UGGGCUGCGGCGUGAAGGU 586 1639-1657 AD-73910 A-147965 CAGCCCCCGGGCCUGGAGA 402 1651-1669 A-147966 UCUCCAGGCCCGGGGGCUG 587 1651-1669 AD-73911 A-147967 GCCUGGAGCGCCUGUGGCU 403 1661-1679 A-147968 AGCCACAGGCGCUCCAGGC 588 1661-1679 AD-73912 A-147969 CUGUGGCUGGAGGGUAACA 404 1672-1690 A-147970 UGUUACCCUCCAGCCACAG 589 1672-1690 AD-73913 A-147971 GGUAACCCCUGGGACUGUA 405 1684-1702 A-147972 UACAGUCCCAGGGGUUACC 590 1684-1702 AD-73914 A-147973 GGGACUGUGGCUGCCCUCU 406 1694-1712 A-147974 AGAGGGCAGCCACAGUCCC 591 1694-1712 AD-73915 A-147975 UGCCCUCUCAAGGCGCUGA 407 1705-1723 A-147976 UCAGCGCCUUGAGAGGGCA 592 1705-1723 AD-73916 A-147977 CGCUGCGGGACUUCGCCCU 408 1718-1736 A-147978 AGGGCGAAGUCCCGCAGCG 593 1718-1736 AD-73917 A-147979 UUCGCCCUGCAGAACCCCA 409 1729-1747 A-147980 UGGGGUUCUGCAGGGCGAA 594 1729-1747 AD-73918 A-147981 CAGAACCCCAGUGCUGUGA 410 1738-1756 A-147982 UCACAGCACUGGGGUUCUG 595 1738-1756 AD-73919 A-147983 UGCUGUGCCCCGCUUCGUA 411 1749-1767 A-147984 UACGAAGCGGGGCACAGCA 596 1749-1767 AD-73920 A-147985 CUUCGUCCAGGCCAUCUGU 412 1761-1779 A-147986 ACAGAUGGCCUGGACGAAG 597 1761-1779 AD-73921 A-147987 CAUCUGUGAGGGGGACGAU 413 1773-1791 A-147988 AUCGUCCCCCUCACAGAUG 598 1773-1791 AD-73922 A-147989 GGGGACGAUUGCCAGCCGA 414 1783-1801 A-147990 UCGGCUGGCAAUCGUCCCC 599 1783-1801 AD-73923 A-147991 CAGCCGCCCGCGUACACCU 415 1795-1813 A-147992 AGGUGUACGCGGGCGGCUG 600 1795-1813 AD-73924 A-147993 CGUACACCUACAACAACAU 416 1805-1823 A-147994 AUGUUGUUGUAGGUGUACG 601 1805-1823 AD-73925 A-147995 AACAACAUCACCUGUGCCA 417 1816-1834 A-147996 UGGCACAGGUGAUGUUGUU 602 1816-1834 AD-73926 A-147997 UGUGCCAGCCCGCCCGAGA 418 1828-1846 A-147998 UCUCGGGCGGGCUGGCACA 603 1828-1846 AD-73927 A-147999 CGCCCGAGGUCGUGGGGCU 419 1838-1856 A-148000 AGCCCCACGACCUCGGGCG 604 1838-1856 AD-73928 A-148001 CGUGGGGCUCGACCUGCGA 420 1848-1866 A-148002 UCGCAGGUCGAGCCCCACG 605 1848-1866 AD-73929 A-148003 ACCUGCGGGACCUCAGCGA 421 1859-1877 A-148004 UCGCUGAGGUCCCGCAGGU 606 1859-1877 AD-73930 A-148005 UCAGCGAGGCCCACUUUGA 422 1871-1889 A-148006 UCAAAGUGGGCCUCGCUGA 607 1871-1889 AD-73931 A-148007 ACUUUGCUCCCUGCUGACA 423 1883-1901 A-148008 UGUCAGCAGGGAGCAAAGU 608 1883-1901 AD-73932 A-148009 CCUGCUGACCAGGUCCCCA 424 1892-1910 A-148010 UGGGGACCUGGUCAGCAGG 609 1892-1910 AD-73933 A-148011 UCCCCGGACUCAAGCCCCA 425 1905-1923 A-148012 UGGGGCUUGAGUCCGGGGA 610 1905-1923 AD-73934 A-148013 CAAGCCCCGGACUCAGGCA 426 1915-1933 A-148014 UGCCUGAGUCCGGGGCUUG 611 1915-1933 AD-73935 A-148015 UCAGGCCCCCACCUGGCUA 427 1927-1945 A-148016 UAGCCAGGUGGGGGCCUGA 612 1927-1945 AD-73936 A-148017 ACCUGGCUCACCUUGUGCU 428 1937-1955 A-148018 AGCACAAGGUGAGCCAGGU 613 1937-1955 AD-73937 A-148019 UUGUGCUGGGGACAGGUCA 429 1949-1967 A-148020 UGACCUGUCCCCAGCACAA 614 1949-1967 AD-73938 A-148021 GACAGGUCCUCAGUGUCCU 430 1959-1977 A-148022 AGGACACUGAGGACCUGUC 615 1959-1977 AD-73939 A-148023 CAGUGUCCUCAGGGGCCUA 431 1969-1987 A-148024 UAGGCCCCUGAGGACACUG 616 1969-1987 AD-73940 A-148025 GGGCCUGCCCAGUGCACUU 432 1981-1999 A-148026 AAGUGCACUGGGCAGGCCC 617 1981-1999 AD-73941 A-148027 UGCACUUGCUGGAAGACGA 433 1993-2011 A-148028 UCGUCUUCCAGCAAGUGCA 618 1993-2011 AD-73942 A-148029 UGGAAGACGCAAGGGCCUA 434 2002-2020 A-148030 UAGGCCCUUGCGUCUUCCA 619 2002-2020 AD-73943 A-148031 AGGGCCUGAUGGGGUGGAA 435 2013-2031 A-148032 UUCCACCCCAUCAGGCCCU 620 2013-2031 AD-73944 A-148033 GGGUGGAAGGCAUGGCGGA 436 2024-2042 A-148034 UCCGCCAUGCCUUCCACCC 621 2024-2042 AD-73945 A-148035 UGGCGGCCCCCCCAGCUGU 437 2036-2054 A-148036 ACAGCUGGGGGGGCCGCCA 622 2036-2054 AD-73946 A-148037 CAGCUGUCAUCAAUUAAAG 438 2048-2066 A-148038 CUUUAAUUGAUGACAGCUG 623 2048-2066 AD-73947 A-148039 AAUUAAAGGCAAAGGCAAU 439 2059-2077 A-148040 AUUGCCUUUGCCUUUAAUU 624 2059-2077 AD-73948 A-148041 AAGGCAAUCGAAUCUAAAA 440 2070-2088 A-148042 UUUUAGAUUCGAUUGCCUU 625 2070-2088

TABLE 7 Human IGFALS Dual-Glo ® in vitro 10 nM screen Duplex Name Average 10 nM STDEV 10 nM AD-73764 46.26 12.94 AD-73765 15.98 9.39 AD-73766 27.71 1.81 AD-73767 29.96 5.64 AD-73768 53.53 15.85 AD-73769 50.94 18.08 AD-73770 35.55 11.71 AD-73771 30.07 11.32 AD-73772 33.23 3.56 AD-73773 11.46 4.14 AD-73774 58.80 12.47 AD-73775 108.20 18.60 AD-73776 51.88 20.74 AD-73777 30.64 7.39 AD-73778 81.00 19.34 AD-73779 78.23 16.91 AD-73780 67.63 20.32 AD-73781 75.04 41.97 AD-73782 11.25 3.14 AD-73783 84.25 27.48 AD-73784 31.16 3.50 AD-73785 40.36 15.91 AD-73786 26.61 4.91 AD-73787 37.73 13.41 AD-73788 41.39 9.64 AD-73789 69.70 17.02 AD-73790 54.70 18.10 AD-73791 37.77 14.31 AD-73792 59.22 4.58 AD-73793 30.72 11.33 AD-73794 96.09 23.63 AD-73795 27.15 4.14 AD-73796 44.57 8.83 AD-73797 22.69 5.07 AD-73798 52.76 11.72 AD-73799 69.71 10.21 AD-73800 49.18 17.49 AD-73801 59.80 17.00 AD-73802 28.96 1.45 AD-73803 33.13 19.76 AD-73804 40.68 7.80 AD-73805 63.69 6.82 AD-73806 66.25 14.80 AD-73807 48.62 17.85 AD-73808 25.07 4.32 AD-73809 68.40 17.86 AD-73810 83.96 14.19 AD-73811 64.13 17.42 AD-73812 46.66 9.77 AD-73813 44.50 17.35 AD-73814 63.89 24.44 AD-73815 52.18 19.16 AD-73816 46.10 24.18 AD-73817 47.24 12.69 AD-73818 26.52 4.62 AD-73819 48.75 11.37 AD-73820 60.19 5.23 AD-73821 94.35 26.80 AD-73822 84.38 36.20 AD-73823 40.82 16.47 AD-73824 73.14 20.30 AD-73825 28.56 4.59 AD-73826 46.85 5.02 AD-73827 47.58 13.90 AD-73828 63.46 15.46 AD-73829 95.35 32.53 AD-73830 58.41 9.47 AD-73831 76.16 9.56 AD-73832 66.65 24.27 AD-73833 48.53 16.86 AD-73834 61.65 17.68 AD-73835 58.15 28.49 AD-73836 26.15 4.79 AD-73837 43.30 9.38 AD-73838 74.76 20.65 AD-73839 78.85 7.72 AD-73840 43.78 12.13 AD-73841 40.30 13.20 AD-73842 43.45 1.12 AD-73843 47.08 8.45 AD-73844 110.22 43.07 AD-73845 53.10 20.78 AD-73846 100.03 52.61 AD-73847 59.82 19.09 AD-73848 26.03 3.83 AD-73849 38.45 7.00 AD-73850 86.08 20.23 AD-73851 61.41 7.67 AD-73852 53.33 19.36 AD-73853 85.67 29.83 AD-73854 54.76 5.66 AD-73855 104.89 36.39 AD-73856 57.24 13.36 AD-73857 63.18 12.14 AD-73858 20.59 3.73 AD-73859 42.26 7.68 AD-73860 94.01 20.91 AD-73861 45.90 18.39 AD-73862 26.77 5.70 AD-73863 39.07 19.21 AD-73864 59.26 14.59 AD-73865 41.82 10.07 AD-73866 60.91 19.05 AD-73867 35.80 9.83 AD-73868 46.58 6.40 AD-73869 64.22 11.51 AD-73870 80.14 7.20 AD-73871 60.16 20.80 AD-73872 56.05 24.26 AD-73873 68.99 18.51 AD-73874 110.04 18.69 AD-73875 45.34 19.36 AD-73876 51.41 17.32 AD-73877 48.52 10.40 AD-73878 114.98 63.70 AD-73879 60.09 8.24 AD-73880 38.19 8.87 AD-73881 74.45 6.60 AD-73882 33.01 9.79 AD-73883 34.58 16.31 AD-73884 53.88 4.17 AD-73885 40.86 12.23 AD-73886 48.81 15.26 AD-73887 100.05 43.02 AD-73888 52.76 9.03 AD-73889 104.07 24.09 AD-73890 34.25 10.25 AD-73891 59.05 17.53 AD-73892 43.11 18.36 AD-73893 74.85 51.34 AD-73894 71.46 42.74 AD-73895 67.51 15.16 AD-73896 65.38 19.16 AD-73897 113.90 19.73 AD-73898 30.88 11.29 AD-73899 71.21 20.59 AD-73900 45.87 8.22 AD-73901 81.14 27.00 AD-73902 57.98 26.64 AD-73903 60.87 50.48 AD-73904 144.84 56.92 AD-73905 80.06 7.93 AD-73906 25.22 6.98 AD-73907 33.52 8.04 AD-73908 88.78 21.09 AD-73909 94.23 19.36 AD-73910 106.31 18.12 AD-73911 64.23 4.10 AD-73912 25.25 5.85 AD-73913 42.38 3.07 AD-73914 38.34 6.64 AD-73915 61.19 28.72 AD-73916 71.86 28.39 AD-73917 95.24 18.35 AD-73918 80.25 27.23 AD-73919 48.91 6.14 AD-73920 39.40 11.01 AD-73921 57.14 12.93 AD-73922 45.90 21.00 AD-73923 56.04 18.98 AD-73924 28.94 7.49 AD-73925 58.43 28.38 AD-73926 102.32 34.13 AD-73927 100.65 27.38 AD-73928 85.51 11.58 AD-73929 51.54 4.93 AD-73930 27.83 6.80 AD-73931 36.71 9.74 AD-73932 37.09 6.54 AD-73933 54.60 14.50 AD-73934 188.17 65.46 AD-73935 77.02 12.48 AD-73936 71.96 24.59 AD-73937 48.37 18.42 AD-73938 47.06 6.65 AD-73939 55.62 19.17 AD-73940 74.83 6.45 AD-73941 41.91 18.36 AD-73942 87.02 43.38 AD-73943 47.56 6.76 AD-73944 39.62 7.36 AD-73945 61.45 10.10 AD-73946 16.22 4.18 AD-73947 17.27 8.22 AD-73948 33.62 7.51

TABLE 8 Modified Sense and Antisense Strand Sequences of IGFALS dsRNAs Sense Anti Anti mRNA Sense Oligo sense sense target Duplex Oligo Se- SEQ Oligo Oligo SEQ Se- SEQ Name Name quence ID Name Seq ID quence ID AD- A- AGGGCA 626 A- UGCCGG 811 AGGGCA  996 73764 147667 GGGGUG 147668 CCACCC GGGGUG GCCGGC CUGCCC GCCGGC AdTdT UdTdT A AD- A- CCGGCA 627 A- UGUACG 812 CCGGCA  997 73765 147669 CAGCAG 147670 UCUGCU CAGCAG ACGUAC GUGCCG ACGUAC AdTdT GdTdT C AD- A- AGACGU 628 A- AGCGAG 813 AGACGU  998 73766 147671 ACCCUC 147672 GGAGGG ACCCUC CCUCGC UACGUC CCUCGC UdTdT UdTdT U AD- A- UCCCUC 629 A- UCAGGC 814 UCCCUC  999 73767 147673 GCUGCC 147674 AGGCAG GCUGCC UGCCUG CGAGGG UGCCUG AdTdT AdTdT C AD- A- UGCCUG 630 A- UCAGGG 815 UGCCUG 1000 73768 147675 CAGCCU 147676 CAGGCU CAGCCU GCCCUG GCAGGC GCCCUG AdTdT AdTdT C AD- A- UGCCCU 631 A- UCAUCC 816 UGCCCU 1001 73769 147677 GCAUGC 147678 UGCAUG GCAUGC AGGAUG CAGGGC AGGAUG AdTdT AdTdT G AD- A- AGGAUG 632 A- CUUUCC 817 AGGAUG 1002 73770 147679 GCCCUG 147680 UCAGGG GCCCUG AGGAAA CCAUCC AGGAAA GdTdT UdTdT G AD- A- CUGAGG 633 A- UCAGGC 818 CUGAGG 1003 73771 147681 AAAGGA 147682 CUCCUU AAAGGA GGCCUG UCCUCA GGCCUG AdTdT GdTdT G AD- A- AGGCCU 634 A- UAGCGC 819 AGGCCU 1004 73772 147683 GGCCCU 147684 CAGGGC GGCCCU GGCGCU CAGGCC GGCGCU AdTdT UdTdT G AD- A- GCGCUG 635 A- AGGACA 820 GCGCUG 1005 73773 147685 CUGCUG 147686 GCAGCA CUGCUG CUGUCC GCAGCG CUGUCC UdTdT CdTdT U AD- A- UGCUGU 636 A- AGUGCC 821 UGCUGU 1006 73774 147687 CCUGGG 147688 ACCCAG CCUGGG UGGCAC GACAGC UGGCAC UdTdT AdTdT U AD- A- UGGCAC 637 A- UUGCGG 822 UGGCAC 1007 73775 147689 UGGGCC 147690 GGGCCC UGGGCC CCCGCA AGUGCC CCCGCA AdTdT AdTdT G AD- A- GCCCCC 638 A- UCCUCC 823 GCCCCC 1008 73776 147691 GCAGCC 147692 AGGCUG GCAGCC UGGAGG CGGGGG UGGAGG AdTdT CdTdT G AD- A- UGGAGG 639 A- UCGGGG 824 UGGAGG 1009 73777 147693 GAGCAG 147694 UCUGCU GAGCAG ACCCCG CCCUCC ACCCCG AdTdT AdTdT G AD- A- AGACCC 640 A- UCCCGG 825 AGACCC 1010 73778 147695 CGGAAC 147696 CGUUCC CGGAAC GCCGGG GGGGUC GCCGGG AdTdT UdTdT G AD- A- CGCCGG 641 A- UCCUCG 826 CGCCGG 1011 73779 147697 GGGAAG 147698 GCUUCC GGGAAG CCGAGG CCCGGC CCGAGG AdTdT GdTdT G AD- A- CGAGGG 642 A- UGGGCA 827 CGAGGG 1012 73780 147699 CCCAGC 147700 CGCUGG CCCAGC GUGCCC GCCCUC GUGCCC AdTdT GdTdT G AD- A- AGCGUG 643 A- ACAGGC 828 AGCGUG 1013 73781 147701 CCCGGC 147702 GGCCGG CCCGGC CGCCUG GCACGC CGCCUG UdTdT UdTdT U AD- A- GCCUGU 644 A- UGUAGC 829 GCCUGU 1014 73782 147703 GUCUGC 147704 UGCAGA GUCUGC AGCUAC CACAGG AGCUAC AdTdT CdTdT G AD- A- UGCAGC 645 A- UGUCGU 830 UGCAGC 1015 73783 147705 UACGAU 147706 CAUCGU UACGAU GACGAC AGCUGC GACGAC AdTdT AdTdT G AD- A- GACGAC 646 A- UGAGCU 831 GACGAC 1016 73784 147707 GCGGAU 147708 CAUCCG GCGGAU GAGCUC CGUCGU GAGCUC AdTdT CdTdT A AD- A- AUGAGC 647 A- UAGAAG 832 AUGAGC 1017 73785 147709 UCAGCG 147710 ACGCUG UCAGCG UCUUCU AGCUCA UCUUCU AdTdT UdTdT G AD- A- UCUUCU 648 A- UUCCUG 833 UCUUCU 1018 73786 147711 GCAGCU 147712 GAGCUG GCAGCU CCAGGA CAGAAG CCAGGA AdTdT AdTdT A AD- A- UCCAGG 649 A- UGCGCG 834 UCCAGG 1019 73787 147713 AACCUC 147714 UGAGGU AACCUC ACGCGC UCCUGG ACGCGC AdTdT AdTdT C AD- A- UCACGC 650 A- UCAUCA 835 UCACGC 1020 73788 147715 GCCUGC 147716 GGCAGG GCCUGC CUGAUG CGCGUG CUGAUG AdTdT AdTdT G AD- A- UGAUGG 651 A- UCCGCC 836 UGAUGG 1021 73789 147717 AGUCCC 147718 CGGGAC AGUCCC GGGCGG UCCAUC GGGCGG AdTdT AdTdT C AD- A- CGGGCG 652 A- AGGGCU 837 CGGGCG 1022 73790 147719 GCACCC 147720 UGGGUG GCACCC AAGCCC CCGCCC AAGCCC UdTdT GdTdT U AD- A- CAAGCC 653 A- UGUCCA 838 CAAGCC 1023 73791 147721 CUGUGG 147722 GCCACA CUGUGG CUGGAC GGGCUU CUGGAC AdTdT GdTdT G AD- A- UGGCUG 654 A- UGUUGU 839 UGGCUG 1024 73792 147723 GACGGC 147724 UGCCGU GACGGC AACAAC CCAGCC AACAAC AdTdT AdTdT C AD- A- AACAAC 655 A- UGACGG 840 AACAAC 1025 73793 147725 CUCUCG 147726 ACGAGA CUCUCG UCCGUC GGUUGU UCCGUC AdTdT UdTdT C AD- A- UCCGUC 656 A- AGGCUG 841 UCCGUC 1026 73794 147727 CCCCCG 147728 CCGGGG CCCCCG GCAGCC GGACGG GCAGCC UdTdT AdTdT U AD- A- CGGCAG 657 A- AGGUUC 842 CGGCAG 1027 73795 147729 CCUUCC 147730 UGGAAG CCUUCC AGAACC GCUGCC AGAACC UdTdT GdTdT U AD- A- CAGAAC 658 A- UCAGGC 843 CAGAAC 1028 73796 147731 CUCUCC 147732 UGGAGA CUCUCC AGCCUG GGUUCU AGCCUG AdTdT GdTdT G AD- A- AGCCUG 659 A- UGUUGA 844 AGCCUG 1029 73797 147733 GGCUUC 147734 GGAAGC GGCUUC CUCAAC CCAGGC CUCAAC AdTdT UdTdT C AD- A- UUCCUC 660 A- UGCCCU 845 UUCCUC 1030 73798 147735 AACCUG 147736 GCAGGU AACCUG CAGGGC UGAGGA CAGGGC AdTdT AdTdT G AD- A- CAGGGC 661 A- UGCCCA 846 CAGGGC 1031 73799 147737 GGCCAG 147738 GCUGGC GGCCAG CUGGGC CGCCCU CUGGGC AdTdT GdTdT A AD- A- AGCUGG 662 A- UGCUCC 847 AGCUGG 1032 73800 147739 GCAGCC 147740 AGGCUG GCAGCC UGGAGC CCCAGC UGGAGC AdTdT UdTdT C AD- A- CUGGAG 663 A- UCAGCG 848 CUGGAG 1033 73801 147741 CCACAG 147742 CCUGUG CCACAG GCGCUG GCUCCA GCGCUG AdTdT GdTdT C AD- A- CGCUGC 664 A- UUCUCU 849 CGCUGC 1034 73802 147743 UGGGCC 147744 AGGCCC UGGGCC UAGAGA AGCAGC UAGAGA AdTdT GdTdT A AD- A- CUAGAG 665 A- UGUGGC 850 CUAGAG 1035 73803 147745 AACCUG 147746 ACAGGU AACCUG UGCCAC UCUCUA UGCCAC AdTdT GdTdT C AD- A- UGUGCC 666 A- UCCAGG 851 UGUGCC 1036 73804 147747 ACCUGC 147748 UGCAGG ACCUGC ACCUGG UGGCAC ACCUGG AdTdT AdTdT A AD- A- ACCUGG 667 A- AGCUGG 852 ACCUGG 1037 73805 147749 AGCGGA 147750 UUCCGC AGCGGA ACCAGC UCCAGG ACCAGC UdTdT UdTdT U AD- A- AACCAG 668 A- UCAGGC 853 AACCAG 1038 73806 147751 CUGCGC 147752 UGCGCA CUGCGC AGCCUG GCUGGU AGCCUG AdTdT UdTdT G AD- A- CGCAGC 669 A- UGCCGA 854 CGCAGC 1039 73807 147753 CUGGCA 147754 GUGCCA CUGGCA CUCGGC GGCUGC CUCGGC AdTdT GdTdT A AD- A- UCGGCA 670 A- UUGUGU 855 UCGGCA 1040 73808 147755 CGUUUG 147756 GCAAAC CGUUUG CACACA GUGCCG CACACA AdTdT AdTdT C AD- A- UUGCAC 671 A- AGCGCG 856 UUGCAC 1041 73809 147757 ACACGC 147758 GGCGUG ACACGC CCGCGC UGUGCA CCGCGC UdTdT AdTdT U AD- A- CCCGCG 672 A- UGAGCG 857 CCCGCG 1042 73810 147759 CUGGCC 147760 AGGCCA CUGGCC UCGCUC GCGCGG UCGCUC AdTdT GdTdT G AD- A- UCGCUC 673 A- UGUUGC 858 UCGCUC 1043 73811 147761 GGCCUC 147762 UGAGGC GGCCUC AGCAAC CGAGCG AGCAAC AdTdT AdTdT A AD- A- AGCAAC 674 A- UGCUCA 859 AGCAAC 1044 73812 147763 AACCGU 147764 GACGGU AACCGU CUGAGC UGUUGC CUGAGC AdTdT UdTdT A AD- A- CUGGAG 675 A- UGAAGA 860 CUGGAG 1045 73813 147767 GACGGG 147768 GCCCGU GACGGG CUCUUC CCUCCA CUCUUC AdTdT GdTdT G AD- A- CUCUUC 676 A- UGCCGA 861 CUCUUC 1046 73814 147769 GAGGGC 147770 GGCCCU GAGGGC CUCGGC CGAAGA CUCGGC AdTdT GdTdT A AD- A- GGCCUC 677 A- UCCAGA 862 GGCCUC 1047 73815 147771 GGCAGC 147772 GGCUGC GGCAGC CUCUGG CGAGGC CUCUGG AdTdT CdTdT G AD- A- UCUGGG 678 A- UCGAGG 863 UCUGGG 1048 73816 147773 ACCUCA 147774 UUGAGG ACCUCA ACCUCG UCCCAG ACCUCG AdTdT AdTdT G AD- A- AACCUC 679 A- UGCUAU 864 AACCUC 1049 73817 147775 GGCUGG 147776 UCCAGC GGCUGG AAUAGC CGAGGU AAUAGC AdTdT UdTdT C AD- A- UGGAAU 680 A- UCACCG 865 UGGAAU 1050 73818 147777 AGCCUG 147778 CCAGGC AGCCUG GCGGUG UAUUCC GCGGUG AdTdT AdTdT C AD- A- CGGUGC 681 A- UCCGCA 866 CGGUGC 1051 73819 147779 UCCCCG 147780 UCGGGG UCCCCG AUGCGG AGCACC AUGCGG AdTdT GdTdT C AD- A- GAUGCG 682 A- UGCCGC 867 GAUGCG 1052 73820 147781 GCGUUC 147782 GGAACG GCGUUC CGCGGC CCGCAU CGCGGC AdTdT CdTdT C AD- A- UUCCGC 683 A- UGCUGC 868 UUCCGC 1053 73821 147783 GGCCUG 147784 CCAGGC GGCCUG GGCAGC CGCGGA GGCAGC AdTdT AdTdT C AD- A- GCAGCC 684 A- ACCAGC 869 GCAGCC 1054 73822 147785 UGCGCG 147786 UCGCGC UGCGCG AGCUGG AGGCUG AGCUGG UdTdT CdTdT U AD- A- GAGCUG 685 A- UGCCCG 870 GAGCUG 1055 73823 147787 GUGCUG 147788 CCAGCA GUGCUG GCGGGC CCAGCU GCGGGC AdTdT CdTdT A AD- A- CUGGCG 686 A- UCAGCC 871 CUGGCG 1056 73824 147789 GGCAAC 147790 UGUUGC GGCAAC AGGCUG CCGCCA AGGCUG AdTdT GdTdT G AD- A- AGGCUG 687 A- UCUGCA 872 AGGCUG 1057 73825 147791 GCCUAC 147792 GGUAGG GCCUAC CUGCAG CCAGCC CUGCAG AdTdT UdTdT C AD- A- ACCUGC 688 A- AAGAGC 873 ACCUGC 1058 73826 147793 AGCCCG 147794 GCGGGC AGCCCG CGCUCU UGCAGG CGCUCU UdTdT UdTdT U AD- A- CGCUCU 689 A- UCCAGG 874 CGCUCU 1059 73827 147795 UCAGCG 147796 CCGCUG UCAGCG GCCUGG AAGAGC GCCUGG AdTdT GdTdT C AD- A- CGGCCU 690 A- UCGGAG 875 CGGCCU 1060 73828 147797 GGCCGA 147798 CUCGGC GGCCGA GCUCCG CAGGCC GCUCCG AdTdT GdTdT G AD- A- AGCUCC 691 A- AGGUCC 876 AGCUCC 1061 73829 147799 GGGAGC 147800 AGCUCC GGGAGC UGGACC CGGAGC UGGACC UdTdT UdTdT U AD- A- CUGGAC 692 A- UGUUCC 877 CUGGAC 1062 73830 147801 CUGAGC 147802 UGCUCA CUGAGC AGGAAC GGUCCA AGGAAC AdTdT GdTdT G AD- A- AGGAAC 693 A- UGGCCC 878 AGGAAC 1063 73831 147803 GCGCUG 147804 GCAGCG GCGCUG CGGGCC CGUUCC CGGGCC AdTdT UdTdT A AD- A- CGGGCC 694 A- UGUUUG 879 CGGGCC 1064 73832 147805 AUCAAG 147806 CCUUGA AUCAAG GCAAAC UGGCCC GCAAAC AdTdT GdTdT G AD- A- AAGGCA 695 A- UCACGA 880 AAGGCA 1065 73833 147807 AACGUG 147808 ACACGU AACGUG UUCGUG UUGCCU UUCGUG AdTdT UdTdT C AD- A- UUCGUG 696 A- UCCGGG 881 UUCGUG 1066 73834 147809 CAGCUG 147810 GCAGCU CAGCUG CCCCGG GCACGA CCCCGG AdTdT AdTdT C AD- A- AGAAAC 697 A- UGGUCC 882 AGAAAC 1067 73835 147813 UCUACC 147814 AGGUAG UCUACC UGGACC AGUUUC UGGACC AdTdT UdTdT G AD- A- CCUGGA 698 A- UAUGAG 883 CCUGGA 1068 73836 147815 CCGCAA 147816 GUUGCG CCGCAA CCUCAU GUCCAG CCUCAU AdTdT GdTdT C AD- A- CCUCAU 699 A- UGCCAC 884 CCUCAU 1069 73837 147817 CGCUGC 147818 GGCAGC CGCUGC CGUGGC GAUGAG CGUGGC AdTdT GdTdT C AD- A- CGUGGC 700 A- UAAGGC 885 CGUGGC 1070 73838 147819 CCCGGG 147820 GCCCGG CCCGGG CGCCUU GGCCAC CGCCUU AdTdT GdTdT C AD- A- GGCGCC 701 A- UCAGGC 886 GGCGCC 1071 73839 147821 UUCCUG 147822 CCAGGA UUCCUG GGCCUG AGGCGC GGCCUG AdTdT CdTdT A AD- A- UGGGCC 702 A- UGCAGC 887 UGGGCC 1072 73840 147823 UGAAGG 147824 GCCUUC UGAAGG CGCUGC AGGCCC CGCUGC AdTdT AdTdT G AD- A- CGCUGC 703 A- AGGUCC 888 CGCUGC 1073 73841 147825 GAUGGC 147826 AGCCAU GAUGGC UGGACC CGCAGC UGGACC UdTdT GdTdT U AD- A- UGGACC 704 A- UGGUUG 889 UGGACC 1074 73842 147827 UGUCCC 147828 UGGGAC UGUCCC ACAACC AGGUCC ACAACC AdTdT AdTdT G AD- A- CACAAC 705 A- UGCCAG 890 CACAAC 1075 73843 147829 CGCGUG 147830 CCACGC CGCGUG GCUGGC GGUUGU GCUGGC AdTdT GdTdT C AD- A- UGGCUG 706 A- UCCUCC 891 UGGCUG 1076 73844 147831 GCCUCC 147832 AGGAGG GCCUCC UGGAGG CCAGCC UGGAGG AdTdT AdTdT A AD- A- CCUGGA 707 A- UGGGAA 892 CCUGGA 1077 73845 147833 GGACAC 147834 CGUGUC GGACAC GUUCCC CUCCAG GUUCCC AdTdT GdTdT C AD- A- UUCCCC 708 A- UGCCCA 893 UUCCCC 1078 73846 147835 GGUCUG 147836 GCAGAC GGUCUG CUGGGC CGGGGA CUGGGC AdTdT AdTdT C AD- A- UGCUGG 709 A- AGCACA 894 UGCUGG 1079 73847 147837 GCCUGC 147838 CGCAGG GCCUGC GUGUGC CCCAGC GUGUGC UdTdT AdTdT U AD- A- CGUGUG 710 A- UGGACA 895 CGUGUG 1080 73848 147839 CUGCGG 147840 GCCGCA CUGCGG CUGUCC GCACAC CUGUCC AdTdT GdTdT C AD- A- CUGUCC 711 A- UGAUGG 896 CUGUCC 1081 73849 147841 CACAAC 147842 CGUUGU CACAAC GCCAUC GGGACA GCCAUC AdTdT GdTdT G AD- A- AACGCC 712 A- UCAGGC 897 AACGCC 1082 73850 147843 AUCGCC 147844 UGGCGA AUCGCC AGCCUG UGGCGU AGCCUG AdTdT UdTdT C AD- A- AGCCUG 713 A- AGGUGC 898 AGCCUG 1083 73851 147845 CGGCCC 147846 GGGGCC CGGCCC CGCACC GCAGGC CGCACC UdTdT UdTdT U AD- A- CGCACC 714 A- UCAGGU 899 CGCACC 1084 73852 147847 UUCAAG 147848 CCUUGA UUCAAG GACCUG AGGUGC GACCUG AdTdT GdTdT C AD- A- AAGGAC 715 A- UCAGGA 900 AAGGAC 1085 73853 147849 CUGCAC 147850 AGUGCA CUGCAC UUCCUG GGUCCU UUCCUG AdTdT UdTdT G AD- A- UUCCUG 716 A- UCUGCA 901 UUCCUG 1086 73854 147851 GAGGAG 147852 GCUCCU GAGGAG CUGCAG CCAGGA CUGCAG AdTdT AdTdT C AD- A- CUGCAG 717 A- UGUUGU 902 CUGCAG 1087 73855 147853 CUGGGC 147854 GGCCCA CUGGGC CACAAC GCUGCA CACAAC AdTdT GdTdT C AD- A- CCACAA 718 A- UUGCCG 903 CCACAA 1088 73856 147855 CCGCAU 147856 GAUGCG CCGCAU CCGGCA GUUGUG CCGGCA AdTdT GdTdT G AD- A- UCCGGC 719 A- UGCUCA 904 UCCGGC 1089 73857 147857 AGCUGG 147858 GCCAGC AGCUGG CUGAGC UGCCGG CUGAGC AdTdT AdTdT G AD- A- UGGCUG 720 A- UCAAAG 905 UGGCUG 1090 73858 147859 AGCGCA 147860 CUGCGC AGCGCA GCUUUG UCAGCC GCUUUG AdTdT AdTdT A AD- A- AGCUUU 721 A- UCCCCA 906 AGCUUU 1091 73859 147861 GAGGGC 147862 GGCCCU GAGGGC CUGGGG CAAAGC CUGGGG AdTdT UdTdT C AD- A- UGGGGC 722 A- AGCACC 907 UGGGGC 1092 73860 147863 AGCUUG 147864 UCAAGC AGCUUG AGGUGC UGCCCC AGGUGC UdTdT AdTdT U AD- A- UUGAGG 723 A- UCUAGC 908 UUGAGG 1093 73861 147865 UGCUCA 147866 GUGAGC UGCUCA CGCUAG ACCUCA CGCUAG AdTdT AdTdT A AD- A- ACGCUA 724 A- UCUGGU 909 ACGCUA 1094 73862 147867 GACCAC 147868 UGUGGU GACCAC AACCAG CUAGCG AACCAG AdTdT UdTdT C AD- A- AACCAG 725 A- UGACCU 910 AACCAG 1095 73863 147869 CUCCAG 147870 CCUGGA CUCCAG GAGGUC GCUGGU GAGGUC AdTdT UdTdT A AD- A- AGGAGG 726 A- UCGCCC 911 AGGAGG 1096 73864 147871 UCAAGG 147872 GCCUUG UCAAGG CGGGCG ACCUCC CGGGCG AdTdT UdTdT C AD- A- CGGGCG 727 A- AGGCCG 912 CGGGCG 1097 73865 147873 CUUUCC 147874 AGGAAA CUUUCC UCGGCC GCGCCC UCGGCC UdTdT GdTdT U AD- A- CUCGGC 728 A- UCACGU 913 CUCGGC 1098 73866 147875 CUCACC 147876 UGGUGA CUCACC AACGUG GGCCGA AACGUG AdTdT GdTdT G AD- A- AACGUG 729 A- UGUUCA 914 AACGUG 1099 73867 147877 GCGGUC 147878 UGACCG GCGGUC AUGAAC CCACGU AUGAAC AdTdT UdTdT C AD- A- UCAUGA 730 A- UUCCCA 915 UCAUGA 1100 73868 147879 ACCUCU 147880 GAGAGG ACCUCU CUGGGA UUCAUG CUGGGA AdTdT AdTdT A AD- A- UCUGGG 731 A- UCCGGA 916 UCUGGG 1101 73869 147881 AACUGU 147882 GACAGU AACUGU CUCCGG UCCCAG CUCCGG AdTdT AdTdT A AD- A- UCUCCG 732 A- UUCCGG 917 UCUCCG 1102 73870 147883 GAACCU 147884 AAGGUU GAACCU UCCGGA CCGGAG UCCGGA AdTdT AdTdT G AD- A- UUCCGG 733 A- UGGAAC 918 UUCCGG 1103 73871 147885 AGCAGG 147886 ACCUGC AGCAGG UGUUCC UCCGGA UGUUCC AdTdT AdTdT G AD- A- GGUGUU 734 A- UCCCAG 919 GGUGUU 1104 73872 147887 CCGGGG 147888 GCCCCG CCGGGG CCUGGG GAACAC CCUGGG AdTdT CdTdT C AD- A- CUGGGC 735 A- UGCUGU 920 CUGGGC 1105 73873 147889 AAGCUG 147890 GCAGCU AAGCUG CACAGC UGCCCA CACAGC AdTdT GdTdT C AD- A- UGCACA 736 A- UCCAGG 921 UGCACA 1106 73874 147891 GCCUGC 147892 UGCAGG GCCUGC ACCUGG CUGUGC ACCUGG AdTdT AdTdT A AD- A- CAGCUG 737 A- UAUGCG 922 CAGCUG 1107 73875 147895 CCUGGG 147896 UCCCAG CCUGGG ACGCAU GCAGCU ACGCAU AdTdT GdTdT C AD- A- GACGCA 738 A- UUGUGC 923 GACGCA 1108 73876 147897 UCCGCC 147898 GGGCGG UCCGCC CGCACA AUGCGU CGCACA AdTdT CdTdT C AD- A- CGCACA 739 A- AGGCCG 924 CGCACA 1109 73877 147899 CCUUCA 147900 GUGAAG CCUUCA CCGGCC GUGUGC CCGGCC UdTdT GdTdT U AD- A- UCACCG 740 A- AGCCCC 925 UCACCG 1110 73878 147901 GCCUCU 147902 GAGAGG GCCUCU CGGGGC CCGGUG CGGGGC UdTdT AdTdT U AD- A- UCGGGG 741 A- AGAGUC 926 UCGGGG 1111 73879 147903 CUCCGC 147904 GGCGGA CUCCGC CGACUC GCCCCG CGACUC UdTdT AdTdT U AD- A- CGACUC 742 A- UGUCCU 927 CGACUC 1112 73880 147905 UUCCUC 147906 UGAGGA UUCCUC AAGGAC AGAGUC AAGGAC AdTdT GdTdT A AD- A- CAAGGA 743 A- UACGAG 928 CAAGGA 1113 73881 147907 CAACGG 147908 GCCGUU CAACGG CCUCGU GUCCUU CCUCGU AdTdT GdTdT G AD- A- GGCCUC 744 A- UCUCAA 929 GGCCUC 1114 73882 147909 GUGGGC 147910 UGCCCA GUGGGC AUUGAG CGAGGC AUUGAG AdTdT CdTdT G AD- A- UUGAGG 745 A- UACAGG 930 UUGAGG 1115 73883 147911 AGCAGA 147912 CUCUGC AGCAGA GCCUGU UCCUCA GCCUGU AdTdT AdTdT G AD- A- AGAGCC 746 A- UCCAGC 931 AGAGCC 1116 73884 147913 UGUGGG 147914 CCCCAC UGUGGG GGCUGG AGGCUC GGCUGG AdTdT UdTdT C AD- A- GGGCUG 747 A- UCAGCA 932 GGGCUG 1117 73885 147915 GCGGAG 147916 GCUCCG GCGGAG CUGCUG CCAGCC CUGCUG AdTdT CdTdT G AD- A- UGCUGG 748 A- UUCAGG 933 UGCUGG 1118 73886 147917 AGCUCG 147918 UCGAGC AGCUCG ACCUGA UCCAGC ACCUGA AdTdT AdTdT C AD- A- GACCUG 749 A- UCUGGU 934 GACCUG 1119 73887 147919 ACCUCC 147920 UGGAGG ACCUCC AACCAG UCAGGU AACCAG AdTdT CdTdT C AD- A- UCCAAC 750 A- UGUGCG 935 UCCAAC 1120 73888 147921 CAGCUC 147922 UGAGCU CAGCUC ACGCAC GGUUGG ACGCAC AdTdT AdTdT C AD- A- ACGCAC 751 A- UGCGGU 936 ACGCAC 1121 73889 147923 CUGCCC 147924 GGGGCA CUGCCC CACCGC GGUGCG CACCGC AdTdT UdTdT C AD- A- CACCGC 752 A- UGCCCU 937 CACCGC 1122 73890 147925 CUCUUC 147926 GGAAGA CUCUUC CAGGGC GGCGGU CAGGGC AdTdT GdTdT C AD- A- UCCAGG 753 A- AGCUUG 938 UCCAGG 1123 73891 147927 GCCUGG 147928 CCCAGG GCCUGG GCAAGC CCCUGG GCAAGC UdTdT AdTdT U AD- A- GCAAGC 754 A- AGCAGG 939 GCAAGC 1124 73892 147929 UGGAGU 147930 UACUCC UGGAGU ACCUGC AGCUUG ACCUGC UdTdT CdTdT U AD- A- UACCUG 755 A- UGCGGG 940 UACCUG 1125 73893 147931 CUGCUC 147932 AGAGCA CUGCUC UCCCGC GCAGGU UCCCGC AdTdT AdTdT A AD- A- CUCUCC 756 A- UCAGGC 941 CUCUCC 1126 73894 147933 CGCAAC 147934 GGUUGC CGCAAC CGCCUG GGGAGA CGCCUG AdTdT GdTdT G AD- A- CCGCCU 757 A- UGGCAG 942 CCGCCU 1127 73895 147935 GGCAGA 147936 CUCUGC GGCAGA GCUGCC CAGGCG GCUGCC AdTdT GdTdT G AD- A- AGCUGC 758 A- AGGGCG 943 AGCUGC 1128 73896 147937 CGGCGG 147938 UCCGCC CGGCGG ACGCCC GGCAGC ACGCCC UdTdT UdTdT U AD- A- GACGCC 759 A- UCAGGG 944 GACGCC 1129 73897 147939 CUGGGC 147940 GGCCCA CUGGGC CCCCUG GGGCGU CCCCUG AdTdT CdTdT C AD- A- CCCCUG 760 A- AGAAGG 945 CCCCUG 1130 73898 147941 CAGCGG 147942 CCCGCU CAGCGG GCCUUC GCAGGG GCCUUC UdTdT GdTdT U AD- A- GGGCCU 761 A- ACGUCC 946 GGGCCU 1131 73899 147943 UCUGGC 147944 AGCCAG UCUGGC UGGACG AAGGCC UGGACG UdTdT CdTdT U AD- A- UGGACG 762 A- UGGUUG 947 UGGACG 1132 73900 147945 UCUCGC 147946 UGCGAG UCUCGC ACAACC ACGUCC ACAACC AdTdT AdTdT G AD- A- ACAACC 763 A- AAUGCC 948 ACAACC 1133 73901 147947 GCCUGG 147948 UCCAGG GCCUGG AGGCAU CGGUUG AGGCAU UdTdT UdTdT U AD- A- GAGGCA 764 A- UGCUGU 949 GAGGCA 1134 73902 147949 UUGCCC 147950 UGGGCA UUGCCC AACAGC AUGCCU AACAGC AdTdT CdTdT C AD- A- CAACAG 765 A- UGGUGC 950 CAACAG 1135 73903 147951 CCUCUU 147952 CAAGAG CCUCUU GGCACC GCUGUU GGCACC AdTdT GdTdT A AD- A- UUGGCA 766 A- UCCGCC 951 UUGGCA 1136 73904 147953 CCACUG 147954 CCAGUG CCACUG GGGCGG GUGCCA GGGCGG AdTdT AdTdT C AD- A- UGGGGC 767 A- AGGUAG 952 UGGGGC 1137 73905 147955 GGCUGC 147956 CGCAGC GGCUGC GCUACC CGCCCC GCUACC UdTdT AdTdT U AD- A- CGCUAC 768 A- UCCUGA 953 CGCUAC 1138 73906 147957 CUCAGC 147958 GGCUGA CUCAGC CUCAGG GGUAGC CUCAGG AdTdT GdTdT A AD- A- UCAGGA 769 A- UGCAGU 954 UCAGGA 1139 73907 147959 ACAACU 147960 GAGUUG ACAACU CACUGC UUCCUG CACUGC AdTdT AdTdT G AD- A- CUCACU 770 A- UGUGAA 955 CUCACU 1140 73908 147961 GCGGAC 147962 GGUCCG GCGGAC CUUCAC CAGUGA CUUCAC AdTdT GdTdT G AD- A- ACCUUC 771 A- UGGGCU 956 ACCUUC 1141 73909 147963 ACGCCG 147964 GCGGCG ACGCCG CAGCCC UGAAGG CAGCCC AdTdT UdTdT C AD- A- CAGCCC 772 A- UCUCCA 957 CAGCCC 1142 73910 147965 CCGGGC 147966 GGCCCG CCGGGC CUGGAG GGGGCU CUGGAG AdTdT GdTdT C AD- A- GCCUGG 773 A- AGCCAC 958 GCCUGG 1143 73911 147967 AGCGCC 147968 AGGCGC AGCGCC UGUGGC UCCAGG UGUGGC UdTdT CdTdT U AD- A- CUGUGG 774 A- UGUUAC 959 CUGUGG 1144 73912 147969 CUGGAG 147970 CCUCCA CUGGAG GGUAAC GCCACA GGUAAC AdTdT GdTdT C AD- A- GGUAAC 775 A- UACAGU 960 GGUAAC 1145 73913 147971 CCCUGG 147972 CCCAGG CCCUGG GACUGU GGUUAC GACUGU AdTdT CdTdT G AD- A- GGGACU 776 A- AGAGGG 961 GGGACU 1146 73914 147973 GUGGCU 147974 CAGCCA GUGGCU GCCCUC CAGUCC GCCCUC UdTdT CdTdT U AD- A- UGCCCU 777 A- UCAGCG 962 UGCCCU 1147 73915 147975 CUCAAG 147976 CCUUGA CUCAAG GCGCUG GAGGGC GCGCUG AdTdT AdTdT C AD- A- CGCUGC 778 A- AGGGCG 963 CGCUGC 1148 73916 147977 GGGACU 147978 AAGUCC GGGACU UCGCCC CGCAGC UCGCCC UdTdT GdTdT U AD- A- UUCGCC 779 A- UGGGGU 964 UUCGCC 1149 73917 147979 CUGCAG 147980 UCUGCA CUGCAG AACCCC GGGCGA AACCCC AdTdT AdTdT A AD- A- CAGAAC 780 A- UCACAG 965 CAGAAC 1150 73918 147981 CCCAGU 147982 CACUGG CCCAGU GCUGUG GGUUCU GCUGUG AdTdT GdTdT C AD- A- UGCUGU 781 A- UACGAA 966 UGCUGU 1151 73919 147983 GCCCCG 147984 GCGGGG GCCCCG CUUCGU CACAGC CUUCGU AdTdT AdTdT C AD- A- CUUCGU 782 A- ACAGAU 967 CUUCGU 1152 73920 147985 CCAGGC 147986 GGCCUG CCAGGC CAUCUG GACGAA CAUCUG UdTdT GdTdT U AD- A- CAUCUG 783 A- AUCGUC 968 CAUCUG 1153 73921 147987 UGAGGG 147988 CCCCUC UGAGGG GGACGA ACAGAU GGACGA UdTdT GdTdT U AD- A- GGGGAC 784 A- UCGGCU 969 GGGGAC 1154 73922 147989 GAUUGC 147990 GGCAAU GAUUGC CAGCCG CGUCCC CAGCCG AdTdT CdTdT C AD- A- CAGCCG 785 A- AGGUGU 970 CAGCCG 1155 73923 147991 CCCGCG 147992 ACGCGG CCCGCG UACACC GCGGCU UACACC UdTdT GdTdT U AD- A- CGUACA 786 A- AUGUUG 971 CGUACA 1156 73924 147993 CCUACA 147994 UUGUAG CCUACA ACAACA GUGUAC ACAACA UdTdT GdTdT U AD- A- AACAAC 787 A- UGGCAC 972 AACAAC 1157 73925 147995 AUCACC 147996 AGGUGA AUCACC UGUGCC UGUUGU UGUGCC AdTdT UdTdT A AD- A- UGUGCC 788 A- UCUCGG 973 UGUGCC 1158 73926 147997 AGCCCG 147998 GCGGGC AGCCCG CCCGAG UGGCAC CCCGAG AdTdT AdTdT G AD- A- CGCCCG 789 A- AGCCCC 974 CGCCCG 1159 73927 147999 AGGUCG 148000 ACGACC AGGUCG UGGGGC UCGGGC UGGGGC UdTdT GdTdT U AD- A- CGUGGG 790 A- UCGCAG 975 CGUGGG 1160 73928 148001 GCUCGA 148002 GUCGAG GCUCGA CCUGCG CCCCAC CCUGCG AdTdT GdTdT G AD- A- ACCUGC 791 A- UCGCUG 976 ACCUGC 1161 73929 148003 GGGACC 148004 AGGUCC GGGACC UCAGCG CGCAGG UCAGCG AdTdT UdTdT A AD- A- UCAGCG 792 A- UCAAAG 977 UCAGCG 1162 73930 148005 AGGCCC 148006 UGGGCC AGGCCC ACUUUG UCGCUG ACUUUG AdTdT AdTdT C AD- A- ACUUUG 793 A- UGUCAG 978 ACUUUG 1163 73931 148007 CUCCCU 148008 CAGGGA CUCCCU GCUGAC GCAAAG GCUGAC AdTdT UdTdT C AD- A- CCUGCU 794 A- UGGGGA 979 CCUGCU 1164 73932 148009 GACCAG 148010 CCUGGU GACCAG GUCCCC CAGCAG GUCCCC AdTdT GdTdT G AD- A- UCCCCG 795 A- UGGGGC 980 UCCCCG 1165 73933 148011 GACUCA 148012 UUGAGU GACUCA AGCCCC CCGGGG AGCCCC AdTdT AdTdT G AD- A- CAAGCC 796 A- UGCCUG 981 CAAGCC 1166 73934 148013 CCGGAC 148014 AGUCCG CCGGAC UCAGGC GGGCUU UCAGGC AdTdT GdTdT C AD- A- UCAGGC 797 A- UAGCCA 982 UCAGGC 1167 73935 148015 CCCCAC 148016 GGUGGG CCCCAC CUGGCU GGCCUG CUGGCU AdTdT AdTdT C AD- A- ACCUGG 798 A- AGCACA 983 ACCUGG 1168 73936 148017 CUCACC 148018 AGGUGA CUCACC UUGUGC GCCAGG UUGUGC UdTdT UdTdT U AD- A- UUGUGC 799 A- UGACCU 984 UUGUGC 1169 73937 148019 UGGGGA 148020 GUCCCC UGGGGA CAGGUC AGCACA CAGGUC AdTdT AdTdT C AD- A- GACAGG 800 A- AGGACA 985 GACAGG 1170 73938 148021 UCCUCA 148022 CUGAGG UCCUCA GUGUCC ACCUGU GUGUCC UdTdT CdTdT U AD- A- CAGUGU 801 A- UAGGCC 986 CAGUGU 1171 73939 148023 CCUCAG 148024 CCUGAG CCUCAG GGGCCU GACACU GGGCCU AdTdT GdTdT G AD- A- GGGCCU 802 A- AAGUGC 987 GGGCCU 1172 73940 148025 GCCCAG 148026 ACUGGG GCCCAG UGCACU CAGGCC UGCACU UdTdT CdTdT U AD- A- UGCACU 803 A- UCGUCU 988 UGCACU 1173 73941 148027 UGCUGG 148028 UCCAGC UGCUGG AAGACG AAGUGC AAGACG AdTdT AdTdT C AD- A- UGGAAG 804 A- UAGGCC 989 UGGAAG 1174 73942 148029 ACGCAA 148030 CUUGCG ACGCAA GGGCCU UCUUCC GGGCCU AdTdT AdTdT G AD- A- AGGGCC 805 A- UUCCAC 990 AGGGCC 1175 73943 148031 UGAUGG 148032 CCCAUC UGAUGG GGUGGA AGGCCC GGUGGA AdTdT UdTdT A AD- A- GGGUGG 806 A- UCCGCC 991 GGGUGG 1176 73944 148033 AAGGCA 148034 AUGCCU AAGGCA UGGCGG UCCACC UGGCGG AdTdT CdTdT C AD- A- UGGCGG 807 A- ACAGCU 992 UGGCGG 1177 73945 148035 CCCCCC 148036 GGGGGG CCCCCC CAGCUG GCCGCC CAGCUG UdTdT AdTdT U AD- A- CAGCUG 808 A- CUUUAA 993 CAGCUG 1178 73946 148037 UCAUCA 148038 UUGAUG UCAUCA AUUAAA ACAGCU AUUAAA GdTdT GdTdT G AD- A- AAUUAA 809 A- AUUGCC 994 AAUUAA 1179 73947 148039 AGGCAA 148040 UUUGCC AGGCAA AGGCAA UUUAAU AGGCAA UdTdT UdTdT U AD- A- AAGGCA 810 A- UUUUAG 995 AAGGCA 1180 73948 148041 AUCGAA 148042 AUUCGA AUCGAA UCUAAA UUGCCU UCUAAA AdTdT UdTdT A

Example 5—In Vitro Screening Cell Culture and Plasmids Transfections for Dual-Glo® Assay:

HeLa cells (ATCC) were transfected by adding 4.9 μl of Opti-MEM plus 0.1 μl of Lipofectamine RNAiMax per well (Invitrogen, Carlsbad Calif. cat #13778-150) to 5 μl of siRNA duplexes per well into a 384-well plate and incubated at room temperature for 15 minutes. Forty μl of Dulbecco's Modified Eagle Medium (Life Tech) containing ˜5×10³ cells were then added to the siRNA mixture. Cells were incubated for 24 hours prior to RNA purification. Single dose experiments were performed at 10 nM and 0.1 nM.

Total RNA Isolation Using DYNABEADS mRNA Isolation Kit

RNA was isolated using an automated protocol on a BioTek-EL406 platform using DYNABEADs (Invitrogen, cat #61012). Briefly, 50 μl of Lysis/Binding Buffer and 25 μl of lysis buffer containing 3 μl of magnetic beads were added to the plate with cells. Plates were incubated on an electromagnetic shaker for 10 minutes at room temperature and then magnetic beads were captured and the supernatant was removed. Bead-bound RNA was then washed 2 times with 150p Wash Buffer A and once with Wash Buffer B. Beads were then washed with 150 μl Elution Buffer, re-captured and supernatant removed.

cDNA Synthesis Using ABI High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, Calif., Cat #4368813)

Ten μl of a master mix containing 1 μl 10× Buffer, 0.4 μl 25×dNTPs, 1 μl 10× Random primers, 0.5 μl Reverse Transcriptase, 0.5 μl RNase inhibitor and 6.6 μl of H2O per reaction was added to RNA isolated above. Plates were sealed, mixed, and incubated on an electromagnetic shaker for 10 minutes at room temperature, followed by 2 hours at 37° C.

Real Time PCR

Two μl of cDNA were added to a master mix containing 0.5 μl of Human GAPDH TaqMan Probe (4326317E), 0.5 μl IGF-1 human probe (Hs01547656 ml) and 5 μl Lightcycler 480 probe master mix (Roche Cat #04887301001) per well in a 384 well plates (Roche cat #04887301001). Real time PCR was done in a LightCycler480 Real Time PCR system (Roche). Each duplex was tested in duplicate and data were normalized to cells transfected with a non-targeting control siRNA.

To calculate relative fold change, real time data were analyzed using the ΔΔCt method and normalized to assays performed with cells transfected with a non-targeting control siRNA.

TABLE 9 Unmodified Sense and Antisense Strand Sequences of IGF-1 dsRNAs Range Range Sense Sense in SEQ Antisense in SEQ Duplex Oligo se- SEQ ID ID Oligo Antisense SEQ ID ID Name Name quence No: 11 NO Name sequence No: 11 NO  AD-66716 A-133440 GCUG 548-568 1181 A-133441 UAUC 546-568 1247 CUUC ACAG CGGA CUCC GCUG GGAA UGAU GCAG A CAC AD-66717 A-133442 UCUG 422-442 1182 A-133443 UCAC 420-442 1248 CGGG CAGC GCUG UCAG AGCU CCCC GGUG GCAG A AGC AD-66718 A-133444 CCUG 378-398 1183 A-133445 UAGC 376-398 1249 CUCA UGGU CCUU GAAG CACC GUGA AGCU GCAG A GCA AD-66719 A-133446 GUGG 461-481 1184 A-133447 AAUA 459-481 1250 AGAC AAAG AGGG CCCC GCUU UGUC UUAU UCCA U CAC AD-66720 A-133448 UGGA 462-482 1185 A-133449 AAAU 460-482 1251 GACA AAAA GGGG GCCC CUUU CUGU UAUU CUCC U ACA AD-66721 A-133450 GAGA 464-484 1186 A-133451 UGAA 462-484 1252 CAGG AUAA GGCU AAGC UUUA CCCU UUUC GUCU A CCA AD-66722 A-133452 CAUG 342-362 1187 A-133453 AAGA 340-362 1253 UCCU GAUG CCUC CGAG GCAU GAGG CUCU ACAU U GGU AD-66723 A-133454 UUUU 475-495 1188 A-133455 UGUG 473-495 1254 AUUU GGCU CAAC UGUU AAGC GAAA CCAC UAAA A AGC AD-66724 A-133456 UGUG 460-480 1189 A-133457 AUAA 458-480 1255 GAGA AAGC CAGG CCCU GGCU GUCU UUUA CCAC U ACA AD-66725 A-133458 UGGA 539-559 1190 A-133459 UCCG 537-559 1256 UGAG GAAG UGCU CAGC GCUU ACUC CCGG AUCC A ACG AD-66726 A-133460 UCGU 455-475 1191 A-133461 AGCC 453-475 1257 GUGU CCUG GGAG UCUC ACAG CACA GGGC CACG U AAC AD-66727 A-133462 GAUG 582-602 1192 A-133463 UUGA 580-602 1258 UAUU GGGG GCGC UGCG ACCC CAAU CUCA ACAU A CUC AD-66728 A-133464 UUCA 449-469 1193 A-133465 UGUC 447-469 1259 GUUC UCCA GUGU CACA GUGG CGAA AGAC CUGA A AGA AD-66729 A-133466 CUCC 348-368 1194 A-133467 AGGU 346-368 1260 UCGC AGAA AUCU GAGA CUUC UGCG UACC AGGA U GGA AD-66730 A-133468 AGAU 581-601 1195 A-133469 UGAG 579-601 1261 GUAU GGGU UGCG GCGC CACC AAUA CCUC CAUC A UCC AD-66731 A-133470 GCCA 638-658 1196 A-133471 UCUU 636-658 1262 CACC GGGC GACA AUGU UGCC CGGU CAAG GUGG A CGC AD-66732 A-133472 GGAG 579-599 1197 A-133473 AGGG 577-599 1263 AUGU GUGC AUUG GCAA CGCA UACA CCCC UCUC U CAG AD-66733 A-133474 UUCA 481-501 1198 A-133475 AUAC 479-501 1264 ACAA CCUG GCCC UGGG ACAG CUUG GGUA UUGA U AAU AD-66734 A-133476 UGCC 630-650 1199 A-133478 AUGU 628-650 1265 CAGC CGGU GCCA GUGG CACC CGCU GACA GGGC U ACG AD-66735 A-133480 GCAU 533-553 1200 A-133482 AGCA 531-553 1266 CGUG GCAC GAUG UCAU AGUG CCAC CUGC GAUG U CCU AD-66736 A-133484 GGAG 463-483 1201 A-133486 UAAA 461-483 1267 ACAG UAAA GGGC AGCC UUUU CCUG AUUU UCUC A CAC AD-66737 A-133488 GGGC 471-491 1202 A-133490 UGCU 469-491 1268 UUUU UGUU AUUU GAAA CAAC UAAA AAGC AGCC A CCU AD-66738 A-133492 UCGU 536-556 1203 A-133494 UGAA 534-556 1269 GGAU GCAG GAGU CACU GCUG CAUC CUUC CACG A AUG AD-66739 A-133496 UCCU 349-369 1204 A-133498 UAGG 347-369 1270 CGCA UAGA UCUC AGAG UUCU AUGC ACCU GAGG A AGG AD-66740 A-133500 GGGG 470-490 1205 A-133502 UCUU 468-490 1271 CUUU GUUG UAUU AAAU UCAA AAAA CAAG GCCC A CUG AD-66741 A-133504 UUUA 476-496 1206 A-133506 UUGU 474-496 1272 UUUC GGGC AACA UUGU AGCC UGAA CACA AUAA A AAG AD-66742 A-133508 GCUG 576-596 1207 A-133510 UGUG 574-596 1273 GAGA CGCA UGUA AUAC UUGC AUCU GCAC CCAG A CCU AD-66743 A-133512 GGGU 496-516 1208 A-133513 UCGA 494-516 1274 AUGG CUGC CUCC UGGA AGCA GCCA GUCG UACC A CUG AD-66744 A-133514 CUGG 577-597 1209 A-133515 UGGU 575-597 1275 AGAU GCGC GUAU AAUA UGCG CAUC CACC UCCA A GCC AD-66745 A-133516 GAAG 330-350 1210 A-133517 UAGG 328-350 1276 AUGC ACAU ACAC GGUG CAUG UGCA UCCU UCUU A CAC AD-66746 A-133477 GAUG 442-462 1211 A-133479 ACAC 440-462 1277 CUCU ACGA UCAG ACUG UUCG AAGA UGUG GCAU U CCA AD-66747 A-133481 UGGA 440-460 1212 A-133483 ACAC 438-460 1278 UGCU GAAC CUUC UGAA AGUU GAGC CGUG AUCC U ACC AD-66748 A-133485 GGUG 438-458 1213 A-133487 ACGA 436-458 1279 GAUG ACUG CUCU AAGA UCAG GCAU UUCG CCAC U CAG AD-66749 A-133489 UGAG 432-452 1214 A-133491 UGAA 430-452 1280 CUGG GAGC UGGA AUCC UGCU ACCA CUUC GCUC A AGC AD-66750 A-133493 GCUG 435-455 1215 A-133495 AACU 433-455 1281 GUGG GAAG AUGC AGCA UCUU UCCA CAGU CCAG U CUC AD-66751 A-133497 GGAU 441-461 1216 A-133499 UACA 439-461 1282 GCUC CGAA UUCA CUGA GUUC AGAG GUGU CAUC A CAC AD-66752 A-133501 CUGG 436-456 1217 A-133503 UAAC 434-456 1283 UGGA UGAA UGCU GAGC CUUC AUCC AGUU ACCA A GCU AD-66753 A-133505 GGCU 429-449 1218 A-133507 AGAG 427-449 1284 GAGC CAUC UGGU CACC GGAU AGCU GCUC CAGC U CCC AD-66754 A-133509 CUGA 431-451 1219 A-133511 UAAG 429-451 1285 GCUG AGCA GUGG UCCA AUGC CCAG UCUU CUCA A GCC AD-66755 A-133518 CAUU 534-554 1220 A-133519 AAGC 532-554 1286 GUGG AACA AUGA CUCA GUGU UCCA UGCU CAAU U GCC AD-66756 A-133520 AGAU * 1221 A-133521 AAGA * 1287 ACAC CGAC AUCA AUGA UGUC UGUG GUCU UAUC U UUU AD-66757 A-133522 UGGA 539-559 1222 A-133523 UCCG 537-559 1288 UGAG GAAG UGUU CAAC GCUU ACUC CCGG AUCC A ACA AD-66758 A-133524 UGUU 547-567 1223 A-133525 AUCA 545-567 1289 GCUU CAGC CCGG UCCG AGCU GAAG GUGA CAAC U ACU AD-66759 A-133526 GCUU 473-493 1224 A-133527 UGGG 471-493 1290 UUAC CUUG UUCA UUGA ACAA AGUA GCCC AAAG A CCC AD-66760 A-133528 AUGA 542-562 1225 A-133529 AGCU 540-562 1291 GUGU CCGG UGCU AAGC UCCG AACA GAGC CUCA U UCC AD-66761 A-133530 CACA 640-660 1226 A-133531 AGUC 638-660 1292 CUGA UUGG CAUG GCAU CCCA GUCA AGAC GUGU U GGC AD-66762 A-133532 GCUA 497-517 1227 A-133533 UCCG 495-517 1293 UGGC AAUG UCCA CUGG GCAU AGCC UCGG AUAG A CCU AD-66763 A-133534 AAGA * 1228 A-133535 AGAC * 1294 UACA GACA CAUC UGAU AUGU GUGU CGUC AUCU U UUA AD-66764 A-133536 UUGC 549-569 1229 A-133537 AGAU 547-569 1295 UUCC CACA GGAG GCUC CUGU CGGA GAUC AGCA U ACA AD-66765 A-133538 UCCG 554-574 1230 A-133539 UCCU 552-574 1296 GAGC CAGA UGUG UCAC AUCU AGCU GAGG CCGG A AAG AD-66766 A-133540 GUGG 538-558 1231 A-133541 UCGG 536-558 1297 AUGA AAGC GUGU AACA UGCU CUCA UCCG UCCA A CAA AD-66767 A-133542 UACA * 1232 A-133543 UUGA * 1298 CAUC AGAC AUGU GACA CGUC UGAU UUCA GUGU A AUC AD-66768 A-133544 AAAG * 1233 A-133545 UACG * 1299 AUAC ACAU ACAU GAUG CAUG UGUA UCGU UCUU A UAU AD-66769 A-133546 GGCU 496-516 1234 A-133547 UCGA 494-516 1300 AUGG AUGC CUCC UGGA AGCA GCCA UUCG UAGC A CUG AD-66770 A-133548 ACAC 641-661 1235 A-133549 UAGU 639-661 1301 UGAC CUUG AUGC GGCA CCAA UGUC GACU AGUG A UGG AD-66771 A-133550 AGUG 545-565 1236 A-133551 UACA 543-565 1302 UUGC GCUC UUCC CGGA GGAG AGCA CUGU ACAC A UCA AD-66772 A-133552 GAGA * 1237 A-133553 UUCA * 1303 CCCU GCCC UUGC CGCA GGGG AAGG CUGA GUCU A CUG AD-66773 A-133554 ACUG 643-663 1238 A-133555 UUGA 641-663 1304 ACAU GUCU GCCC UGGG AAGA CAUG CUCA UCAG A UGU AD-66774 A-133556 GAUA * 1239 A-133557 UAAG * 1305 CACA ACGA UCAU CAUG GUCG AUGU UCUU GUAU A CUU AD-66775 A-133558 AAGC 487-507 1240 A-133559 UGAG 485-507 1306 CCAC CCAU AGGC AGCC UAUG UGUG GCUC GGCU A UGU  Range Sense Sense in SEQ Antisense Range in SEQ Duplex Oligo se- SEQ ID ID Oligo Antisense SEQ ID ID Name Name quence No: 17 NO Name sequence No: 17 NO  AD-66756 A-133520 AGAU 366-368 1241 A-133521 AAGA 364-368 1307 ACAC CGAC AUCA AUGA UGUC UGUG GUCU UAUC U UUU AD-66763 A-133534 AAGA 365-385 1242 A-133535 AGAC 363-385 1308 UACA GACA CAUC UGAU AUGU GUGU CGUC AUCU U UUA AD-66767 A-133542 UACA 369-389 1243 A-133543 UUGA 367-389 1309 CAUC AGAC AUGU GACA CGUC UGAU UUCA GUGU A AUC AD-66768 A-133544 AAAG 364-384 1244 A-133545 UACG 362-384 1310 AUAC ACAU ACAU GAUG CAUG UGUA UCGU UCUU A UAU AD-66772 A-133552 GAGA 449-469 1245 A-133553 UUCA 447-469 1311 CCCU GCCC UUGC CGCA GGGG AAGG CUGA GUCU A CUG AD-66774 A-133556 GAUA 367-387 1246 A-133557 UAAG 365-387 1312 CACA ACGA UCAU CAUG GUCG AUGU UCUU GUAU A CUU  * Targeting sequence in NNI_010512 (SEQ ID NO: 7).

TABLE 10 IGF-1 Screen in HeLa cells Each duplex was tested in duplicate and data were normalized to cells transfected with a non-targeting control siRNA AD-1955. Duplex Name 10 nM Avg STDEV 0.1 nM Avg STDEV AD-66716 58.4 3.1 90.3 24.0 AD-66717 82.2 1.6 81.1 10.3 AD-66718 62.7 7.0 76.4 10.1 AD-66719 51.0 6.7 83.2 8.1 AD-66720 30.3 0.9 74.9 15.7 AD-66721 58.0 8.5 82.5 23.9 AD-66722 4.7 1.0 29.2 9.0 AD-66723 70.5 7.3 85.4 11.3 AD-66724 32.4 8.0 82.7 9.7 AD-66725 22.6 6.4 72.8 12.1 AD-66726 32.8 2.1 76.4 25.0 AD-66727 53.5 1.1 83.1 6.5 AD-66728 59.1 11.5 91.5 15.2 AD-66729 27.9 4.2 75.3 27.4 AD-66730 79.1 12.7 87.8 18.4 AD-66731 86.4 15.9 97.0 25.4 AD-66732 81.0 8.3 80.9 15.0 AD-66733 8.7 3.7 43.7 1.9 AD-66734 65.4 3.2 83.7 15.5 AD-66735 62.0 6.7 82.6 8.1 AD-66736 71.9 4.5 83.5 23.4 AD-66737 68.7 4.0 92.0 14.4 AD-66738 19.2 3.3 79.4 14.3 AD-66739 10.6 2.7 61.8 23.6 AD-66740 23.2 4.4 68.2 14.6 AD-66741 83.6 0.5 76.5 14.2 AD-66742 73.1 2.2 86.2 10.1 AD-66743 58.9 1.8 88.1 11.2 AD-66744 53.9 2.1 96.8 7.6 AD-66745 28.3 5.5 76.8 7.9 AD-66746 6.3 0.7 50.1 3.9 AD-66747 8.5 2.8 50.0 0.0 AD-66748 6.2 1.3 34.8 4.1 AD-66749 30.0 0.5 92.4 3.2 AD-66750 27.6 0.7 74.8 1.5 AD-66751 50.1 0.3 89.3 15.2 AD-66752 9.6 1.3 55.5 12.4 AD-66753 54.6 2.1 89.0 0.9 AD-66754 78.6 16.8 104.3 2.0 AD-66755 46.8 4.8 103.4 18.6 AD-66756 86.3 2.5 95.9 18.2 AD-66757 69.1 0.7 103.0 5.0 AD-66758 67.5 3.6 86.5 2.5 AD-66759 106.5 16.2 91.4 20.4 AD-66760 54.2 1.6 86.8 0.4 AD-66761 40.8 3.8 92.2 7.2 AD-66762 96.8 7.1 100.6 8.4 AD-66763 81.2 11.4 92.1 0.0 AD-66764 86.0 2.2 101.2 12.9 AD-66765 100.9 13.7 93.2 25.3 AD-66766 36.6 3.9 78.5 15.7 AD-66767 124.0 12.2 89.9 1.3 AD-66768 113.9 15.1 92.7 15.4 AD-66769 92.0 7.1 93.6 8.2 AD-66770 79.7 3.6 98.7 1.9 AD-66771 60.6 12.1 97.6 10.5 AD-66772 95.5 7.0 95.9 9.9 AD-66773 61.3 3.9 90.7 7.5 AD-66774 95.6 8.9 81.9 21.8 AD-66775 113.1 13.9 99.5 6.8 AD-1955 100.0 8.0

TABLE 11 Modified Sense and Antisense Sequences of IGF-1 Sense Modified  SEQ Antisense Modified SEQ Duplex  Oligo Sense ID Oligo Antisense ID Name Name Sequence NO Name Sequence NO: AD- A- GfscsU 1313 A- usAfsu 1373 66716 133440 fgCfuU 133441 CfaCfa fcCfGf GfcUfc GfaGfc cgGfaA UfgUfg fgCfaG AfuAfL fcsasc 96 AD- A- UfscsU 1314 A- usCfsa 1374 66717 133442 fgCfgG 133443 CfcAfg fgGfCf CfuCfa UfgAfg gcCfcC CfuGfg fgCfaG UfgAfL fasgsc 96 AD- A- CfscsU 1315 A- usAfsg 1375 66718 133444 fgCfuC 133445 CfuGfg faCfCf UfgAfa UfuCfa ggUfgA CfcAfg fgCfaG CfuAfL fgscsa 96 AD- A- GfsusG 1316 A- asAfsu 1376 66719 133446 fgAfgA 133447 AfaAfa fcAfGf GfcCfc GfgGfc cuGfuC UfuUfu fuCfcA AfuUfL fcsasc 96 AD- A- UfsgsG 1317 A- asAfsa 1377 66720 133448 faGfaC 133449 UfaAfa faGfGf AfgCfc GfgCfu ccUfgU UfuUfa kUfcCf UfuUfL ascsa 96 AD- A- GfsasG 1318 A- usGfsa 1378 66721 133450 faCfaG 133451 AfaUfa fgGfGf AfaAfg CfuUfu ccCfcU UfaUfu fgUfcU UfcAfL lcscsa 96 AD- A- CfsasU 1319 A- asAfsg 1379 66722 133452 fgUfcC 133453 AfgAfu fuCfCf GfcGfa UfcGfc ggAfgG AfuCfu faCfaU CfuUfL fgsgsu 96 AD- A- UfsusU 1320 A- usGfsu 1380 66723 133454 fuAfuU 133455 GfgGfc fuCfAf UfuGfu AfcAfa ugAfaA GfcCfc fuAfaA AfcAfL fasgsc 96 AD- A- UfsgsU 1321 A- asUfsa 1381 66724 133456 fgGfaG 133457 AfaAfg faCfAf CfcCfc GfgGfg ugUfal CfuUfu fcCfaC UfaUfL fascsa 96 AD- A- UfsgsG 1322 A- usCfsc 1382 66725 133458 faUfgA 133459 GfgAfa fgUfGf GfcAfg CfuGfc caCfuC UfuCfc faUfcC GfgAfL fascsg 96 AD- A- UfscsG 1323 A- asGfsc 1383 66726 133460 fuGfuG 133461 CfcCfu fuGfGf GfuCfu AfgAfc ccAfcA AfgGfg fcAfcG GfcUfL fasasc 96 AD- A- GfsasU 1324 A- usUfsg 1384 66727 133462 fgUfaU 133463 AfgGfg fuGfCf GfuGfc GfcAfc gcAfaU CfcCfu faCfaU CfaAfL fcsusc 96 AD- A- UfsusC 1325 A- usGfsu 1385 66728 133464 faGfuU 133465 CfuCfc fcGfUf AfcAfc GfuGfu acGfaA GfgAfg fcUfgA AfcAfL fasgsa 96 AD- A- CfsusC 1326 A- asGfsg 1386 66729 133466 fcUfcG 133467 UfaGfa fcAfUf AfgAfg CfuCfu auGfcG UfcUfa faGfgA CfcUfL fgsgsa 96 AD- A- AfsgsA 1327 A- usGfsa 1387 66730 133468 fuGfuA 133469 GfgGfg fuUfGf UfgCfg CfgCfa caAfuA CfcCfc fcAfuC UfcAfL fuscsc 96 AD- A- GfscsC 1328 A- usCfsu 1388 66731 133470 faCfaC 133471 UfgGfg fcGfAf CfaUfg CfaUfg ucGfgU CfcCfa fgUfgG AfgAfL fcsgsc 96 AD- A- GfsgsA 1329 A- asGfsg 1389 66732 133472 fgAfuG 133473 GfgUfg fuAfUf CfgCfa UfgCfg auAfcA CfaCfc fuCfuC CfcUfL fcsasg 96 AD- A- UfsusC 1330 A- asUfsa 1390 66733 133474 faAfcA 133475 CfcCfu faGfCf GfuGfg CfcAfc gcUfuG AfgGfg fuUfgA UfaUfL fasasu 96 AD- A- UfsgsC 1331 A- asUfsg 1391 66734 133476 fcCfaG 133478 UfcGfg fcGfCf UfgUfg CfaCfa gcGfcU CfcGfa fgGfgC CfaUfL fascsg 96 AD- A- GfscsA 1332 A- asGfsc 1392 66735 133480 fuCfgU 133482 AfgCfa fgGfAf CfuCfa UfgAfg ucCfaC UfgCfu fgAfuG GfcUfL fcscsu 96 AD- A- GfsgsA 1333 A- usAfsa 1393 66736 133484 fgAfcA 133486 AfuAfa fgGfGf AfaGfc GfcUfu ccCfuG UfuAfu fuCfuC UfuAfL fcsasc 96 AD- A- GfsgsG 1334 A- usGfsc 1394 66737 133488 fcUfuU 133490 UfuGfu fuAfUf UfgAfa UfuCfa auAfaA AfcAfa faGfcC GfcAfL fcscsu 96 AD- A- UfscsG 1335 A- usGfsa 1395 66738 133492 fuGfgA 133494 AfgCfa fuGfAf GfcAfc GfuGfc ucAfuC UfgCfu fcAfcG UfcAfL fasusg 96 AD- A- UfscsC 1336 A- usAfsg 1396 66739 133496 fuCfgC 133498 GfuAfg faUfCf AfaGfa Ufalfu gaUfgC CfuAfc fgAfgG CfuAfL fasgsg 96 AD- A- GfsgsG 1337 A- usCfsu 1397 66740 133500 fgCfuU 133502 UfgUfu fuUfAf GfaAfa UfuUfc uaAfaA AfaCfa fgCfcC AfgAfL fcsusg 96 AD- A- UfsusU 1338 A- usUfsg 1398 66741 133504 faUfuU 133506 UfgGfg fcAfAf CfuUfg CfaAfg uuGfaA CfcCfa faUfaA CfaAfL fasasg 96 AD- A- GfscsU 1339 A- usGfsu 1399 66742 133508 fgGfaG 133510 GfcGfc faUfGf AfaUfa UfaUfu caUfcU GfcGfc fcCfaG AfcAfL fcscsu 96 AD- A- GfsgsG 1340 A- usCfsg 1400 66743 133512 fuAfuG 133513 AfcUfg fgCfUf CfuGfg CfcAfg agCfcA CfaGfu fuAfcC CfgAfL fcsusg 96 AD- A- CfsusG 1341 A- usGfsg 1401 66744 133514 fgAfgA 133515 UfgCfg fuGfUf CfaAfu AfuUfg acAfuC CfgCfa fuCfcA CfcAfL fgscsc 96 AD- A- GfsasA 1342 A- usAfsg 1402 66745 133516 fgAfuG 133517 GfaCfa fcAfCf UfgGfu AfcCfa guGfcA UfgUfc fuCfuU CfuAfL fcsasc 96 AD- A- GfsasU 1343 A- asCfsa 1403 66746 133477 fgCfuC 133479 CfaCfg fuUfCf AfaCfu AfgUfu gaAfgA CfgUfg fgCfaU UfgUfL fcscsa 96 AD- A- UfsgsG 1344 A- asCfsa 1404 66747 133481 faUfgC 133483 CfgAfa fuCfUf CfuGfa UfcAfg agAfgC UfuCfg faUfcC UfgUfL fascsc 96 AD- A- GfsgsU 1345 A- asCfsg 1405 66748 133485 fgGfaU 133487 AfaCfu fgCfUf GfaAfg CfuUfc agCfaU AfgUfu fcCfaC CfgUfL fcsasg 96 AD- A- UfsgsA 1346 A- usGfsa 1406 66749 133489 fgCfuG 133491 AfgAfg fgUfGf CfaUfc GfaUfg caCfcA CfuCfu fgCfuC UfcAfL fasgsc 96 AD- A- GfscsU 1347 A- asAfsc 1407 66750 133493 fgGfuG 133495 UfgAfa fgAfUf GfaGfc GfcUfc auCfcA UfuCfa fcCfaG GfuUfL fcsusc 96 AD- A- GfsgsA 1348 A- usAfsc 1408 66751 133497 fuGfcU 133499 AfcGfa fcUfUf AfcUfg CfaGfu aaGfaG UfcGfu fcAfuC GfuAfL fcsasc 96 AD- A- CfsusG 1349 A- usAfsa 1409 66752 133501 fgUfgG 133503 CfuGfa faUfGf AfgAfg CfuCfu caUfcC UfcAfg faCfcA UfuAfL fgscsu 96 AD- A- GfsgsC 1350 A- asGfsa 1410 66753 133505 fuGfaG 133507 GfcAfu fcUfGf CfcAfc GfuGfg caGfcU AfuGfc fcAfgC UfcUfL fcscsc 96 AD- A- CfsusG 1351 A- usAfsa 1411 66754 133509 faGfcU 133511 GfaGfc fgGfUf AfuCfc GfgAfu acCfaG GfcUfc fcUfcA UfuAfL fgscsc 96 AD- A- CfsasU 1352 A- asAfsg 1412 66755 133518 fuGfuG 133519 CfaAfc fgAfUf AfcUfc GfaGfu auCfcA GfuUfg fcAfaU CfuUfL fgscsc 96 AD- A- AfsgsA 1353 A- asAfsg 1413 66756 133520 fuAfcA 133521 AfcGfa fcAfUf CfaUfg CfaUfg auGfuG UfcGfu fuAfuC CfuUfL fususu 96 AD- A- UfsgsG 1354 A- usCfsc 1414 66757 133522 faUfgA 133523 GfgAfa fgUfGf GfcAfa UfuGfc caCfuC UfuCfc faUfcC GfgAfL fascsa 96 AD- A- UfsgsU 1355 A- asUfsc 1415 66758 133524 fuGfcU 133525 AfcAfg fuCfCf CfuCfc GfgAfg ggAfaG CfuGfu fcAfaC GfaUfL fascsu 96 AD- A- GfscsU 1356 A- usGfsg 1416 66759 133526 fuUfuA 133527 GfcUfu fcUfUf GfuUfg CfaAfc aaGfuA AfaGfc faAfaG CfcAfL fcscsc 96 AD- A- AfsusG 1357 A- asGfsc 1417 66760 133528 faGfuG 133529 UfcCfg fuUfGf GfaAfg CfuUfc caAfcA CfgGfa fcUfcA GfcUfL fuscsc 96 AD- A- CfsasC 1358 A- asGfsu 1418 66761 133530 faCfuG 133531 CfuUfg faCfAf GfgCfa UfgCfc ugUfcA CfaAfg fgUfgU AfcUfL fgsgsc 96 AD- A- GfscsU 1359 A- usCfsc 1419 66762 133532 faUfgG 133533 GfaAfu fcUfCf GfcUfg CfaGfc gaGfcC AfuUfc faUfaG GfgAfL fcscsu 96 AD- A- AfsasG 1360 A- asGfsa 1420 66763 133534 faUfaC 133535 CfgAfc faCfAf AfuGfa UfcAfu ugUfgU GfuCfg faUfcU UfcUfL fususa 96 AD- A- UfsusG 1361 A- asGfsa 1421 66764 133536 fcUfuC 133537 UfcAfc fcGfGf AfgCfu AfgCfu ccGfgA GfuGfa faGfcA UfcUfL fascsa 96 AD- A- UfscsC 1362 A- usCfsc 1422 66765 133538 fgGfaG 133539 UfcAfg fcUfGf AfuCfa UfgAfu caGfcU CfuGfa fcCfgG GfgAfL fasasg 96 AD- A- GfsusG 1363 A- usCfsg 1423 66766 133540 fgAfuG 133541 GfaAfg faGfUf CfaAfc GfuUfg acUfcA CfuUfc fuCfcA CfgAfL fcsasa 96 AD- A- UfsasC 1364 A- usUfsg 1424 66767 133542 faCfaU 133543 AfaGfa fcAfUf CfgAfc GfuCfg auGfaU UfcUfu fgUfgU CfaAfL fasusc 96 AD- A- AfsasA 1365 A- usAfsc 1425 66768 133544 fgAfuA 133545 GfaCfa fcAfCf UfgAfu AfuCfa guGfuA UfgUfc fuCfuU GfuAfL fusasu 96 AD- A- GfsgsC 1366 A- usCfsg 1426 66769 133546 fuAfuG 133547 AfaUfg fgCfUf CfuGfg CfcAfg agCfcA CfaUfu fuAfgC CfgAfL fcsusg 96 AD- A- AfscsA 1367 A- usAfsg 1427 66770 133548 fcUfgA 133549 UfcUfu fcAfUf GfgGfc GfcCfc auGfuC AfaGfa faGfuG CfuAfL fusgsg 96 AD- A- AfsgsU 1368 A- usAfsc 1428 66771 133550 fgUfuG 133551 AfgCfu fcUfUf CfcGfg CfcGfg aaGfcA AfgCfu faCfaC GfuAfL fuscsa 96 AD- A- GfsasG 1369 A- usUfsc 1429 66772 133552 faCfcC 133553 AfgCfc fuUfUf CfcGfc GfcGfg aaAfgG GfgCfu fgUfcU GfaAfL lcsusg 96 AD- A- AfscsU 1370 A- usUfsg 1430 66773 133554 fgAfcA 133555 AfgUfc fuGfCf UfuGfg CfcAfa gcAfuG GfaCfu fuCfaG CfaAfL fusgsu 96 AD- A- GfsasU 1371 A- usAfsa 1431 66774 133556 faCfaC 133557 GfaCfg faUfCf AfcAfu AfuGfu gaUfgU CfgUfc fgUfaU UfuAfL fcsusu 96 AD- A- AfsasG 1372 A- usGfsa 1432 66775 133558 fcCfcA 133559 GfcCfa fcAfGf UfaGfc GfcUfa cuGfuG UfgGfc fgGfcU UfcAfL fusgsu 96

Example 6—Knockdown of IGF-1 Expression with an IGF-1 siRNA Decreases Expression of IGF-1

A series of siRNAs targeting mouse IGF-1 were designed and tested for the ability to knockdown expression of IGF-1 mRNA in 6-8 week old C57Bl/6 female mice. Duplexes were selected for further optimization using chemical modifications. Analysis for IGF-1 knockdown in mice using the same assay identified the AD-68112 duplex (sense sequence gsasuacaCfaUfCfAfugucgucuuaL96 (SEQ ID NO: 1433); antisense sequence usAfsagaCfgAfCfaugaUfgUfguaucsusu (SEQ ID NO: 1434), based on the sequence of the AD-66774 duplex, for use in further studies.

A single 3 mg/kg or 10 mg/kg dose of AD-68112; or PBS control, was administered subcutaneously on day 0 to 6-8 week old C57Bl/6 female mice (n=3 per group). On days 7, 14, and 21 the mice were sacrificed to assess knockdown of IGF-1 mRNA in liver by qPCR.

AD-68112 was found to be effective in decreasing expression of IGF-1 mRNA. The results are shown in the table below. Results are expressed as mRNA levels relative to control.

Dose Day 7 Day 14 Day 21  3 mg/kg 7.4 17.8 33.3 10 mg/kg 3.4 7.4 12.7

In a separate study, a decrease in serum IGF-1 was observed in 6-8 week old C57Bl/6 female mice (n=3) response to treatment with AD-68112 in a single dose of 3 mg/kg. Specifically, AD-68112 decreased the serum IGF-1 protein level to about 17%, 70%, and 55% on days 7, 14, and 21, respectively.

Further, in a dose-response study, AD-68112 was demonstrated to be effective in knocking down the expression of IGF-1 mRNA in the liver in a dose-response manner. Specifically, C57Bl/6 female mice, 6-8 weeks of age (n=3 per group) were administered a single 0.3 mg/kg, 1 mg/kg, 3 mg/kg, or 10 mg/kg AD-68112 at; or a PBS control. IGF-1 serum protein level reduction was observed in a dose response manner.

Example 7—Knockdown of IGFALS and IGF-1 Expression with IGFALS and IGF-1 siRNAs Alone and in Combination

A series of siRNAs targeted each to mouse IGFALS (AD-66807, sense sequence, ascsagauGfaGfCfUfcagcgucuuuL96, SEQ ID NO: 1435; and antisense sequence asAfsagaCfgCfUfgagcUfcAfucugusgsu, SEQ ID NO: 1436) and mouse IGF-1 (AD-68112) were tested for the ability to knockdown expression of IGFALS and IGF-1 mRNA and protein expression in 6-8 week old C57Bl/6 female mice, either alone or in combination.

Weekly 3 mg/kg doses of AD-66807 and AD-68112, either alone or in combination; or PBS control, were administered to 4 week old C57Bl/6 female mice (n=8 per group) subcutaneously starting at day 0 for 8 weeks. On day 58 or 59, the mice were sacrificed to assess knockdown of IGFALS and IGF-1 mRNA in liver by qPCR. Serum IGFALS levels were assayed by western blot and serum IGF-1 levels were assayed by ELISA.

AD-66807 and AD-68112 were found to be effective in decreasing expression of IGFALS and IGF-1 mRNA and protein. The results are shown in the tables below. RNA and protein levels are expressed as levels relative to control.

IGFALS and IGF-1 mRNA Levels Relative to Control

Treatment IGFALS IGF-1 Control (PBS) 1.16 1.05 SiRNA-IGFALS (3 mg/kg) 0.11 1.10 SiRNA-IGF-1 (3 mg/kg) 0.70 0.03 SiRNA-ALS (3 mg/kg) + IGF-1 (3 mg/kg) 0.09 0.05

IGFALS and IGF-1 Protein Levels Relative to Control.

Treatment IGFALS IGF-1 Control (PBS) 1.0 1.0 SiRNA-IGFALS (3 mg/kg) 0.03 0.26 SiRNA-IGF-1 (3 mg/kg) 0.61 0.13 SiRNA-ALS (3 mg/kg) + IGF-1 (3 mg/kg) 0.01 0.05

A dose response study was performed. AD-66807 and AD-68112 were subcutaneously administered to 6-8 week old C57Bl/6 female mice (n=3 per group) at 0.3 mg/kg, 1 mg/kg, 3 mg/kg, and 10 mg/kg, either alone or in combination; or PBS control starting at day 0. On day 14, the mice were sacrificed to assess knockdown of IGFALS and IGF-1 mRNA in liver by qPCR. Serum IGF-1 levels were assayed by ELISA.

AD-66807 and AD-68112 were found to be effective in decreasing expression of serum IGF-1 protein. The results are shown in the table below. Protein levels are expressed as levels relative to control.

Serum IGF-1 Protein Levels Relative to Control at Day 7.

0 0.3 1.0 3.0 10.0 Treatment mg/kg mg/kg mg/kg mg/kg mg/kg SiRNA-IGFALS 100 0.81 0.54 0.37 0.31 SiRNA-IGF-1 100 1.13 0.83 0.52 0.49 SiRNA-IGFALS + 100 0.61 0.34 0.14 0.06 SiRNA-IGF-1

Example 8—Knockdown of IGFALS and IGF-1 Expression with IGFALS and IGF-1 siRNAs Alone and in Combination in a Transgenic Mouse Expressing Bovine Growth Hormone

Similar knockdown of serum IGFALS and IGF-1 levels were observed in a transgenic mouse that constitutively expresses bovine growth hormone and recapitulates some of the features of acromegaly (Olsson et al, Am J Physiol Endocrinol Metab. 2003; 285:E504-11). Specifically, AD-66807 and AD-68112 were demonstrated to decrease serum levels of IGF-1 protein. At least a trend in decreased weight gain was observed in male mice treated with either AD-66807 or AD-68112 alone, and in female mice treated with a combination of AD-66807 and AD-68112.

Example 9—IGFALS Transcripts, siRNA Design, and siRNA Screening

A set of siRNAs targeting the human IGFALS, “insulin like growth factor binding protein acid labile subunit” (human: NCBI refseqID NM_004970 (SEQ ID NO: 1); NCBI GeneID: 3483), as well as toxicology-species IGFALS orthologs (cynomolgus monkey: XM_005590898) were designed using custom R and Python scripts. The human NM_004970 REFSEQ mRNA, version 2, has a length of 2168 bases.

The rationale and method for the set of siRNA designs is as follows: the predicted efficacy for every potential 19mer siRNA from position 10 through the end was determined with a linear model derived the direct measure of mRNA knockdown from more than 20,000 distinct siRNA designs targeting a large number of vertebrate genes. Subsets of the IGFALS siRNAs were designed with perfect or near-perfect matches between human and cynomolgus monkey. For each strand of the siRNA, a custom Python script was used in a brute force search to measure the number and positions of mismatches between the siRNA and all potential alignments in the target species transcriptome. Extra weight was given to mismatches in the seed region, defined here as positions 2-9 of the antisense oligonucleotide, as well the cleavage site of the siRNA, defined here as positions 10-11 of the antisense oligonucleotide. The relative weight of the mismatches was 2.8; 1.2:1 for seed mismatches, cleavage site, and other positions up through antisense position 19. Mismatches in the first position were ignored. A specificity score was calculated for each strand by summing the value of each weighted mismatch. Preference was given to siRNAs whose antisense score in human was >=2.2 and predicted efficacy was >=50% knockdown of the transcript.

In Vitro Dual-Glo® Screening

Cell Culture and Transfections

Cos7 cells (ATCC, Manassas, Va.) were grown to near confluence at 37° C. in an atmosphere of 5% CO₂ in DMEM (ATCC) supplemented with 10% FBS, before being released from the plate by trypsinization. Human IGFALS (NM 004970 (SEQ ID NO: 1) was cloned into the psicheck2 vector to generate the Dual-Glo® Luciferase construct. The Dual-luciferase plasmid was co-transfected with siRNA into 5000 cells using Lipofectamine RNAiMax (Invitrogen, Carlsbad Calif. cat #13778-150). For each well of a 384 well plate, 0.1 μl of Lipofectamine was added to 5 ng of plasmid vector and siRNA in 15 μl of Opti-MEM and allowed to complex at room temperature for 15 minutes. The mixture was then added to the cells resuspended in 35 ul of fresh complete media. Cells were incubated for 48 hours before luciferase was measured. Screens were performed at 10 nM and 0.1 nM final duplex concentration.

Dual-Glo® Luciferase Assay

Forty-eight hours after the siRNAs were transfected, Firefly (transfection control) and Renilla (fused to IGFALS target sequence in 3′ UTR) luciferase were measured. First, media was removed from cells. Then Firefly luciferase activity was measured by adding 20 ul of Dual-Glo® Luciferase Reagent mixed with 20 ul of complete media to each well. The mixture was incubated at room temperature for 30 minutes before luminescense (500 nm) was measured on a Spectramax (Molecular Devices) to detect the Firefly luciferase signal. Renilla luciferase activity was measured by adding 20 ul of room temperature of Dual-Glo® Stop & Glo® Reagent to each well and the plates were incubated for 20 minutes before luminescence was again measured to determine the Renilla luciferase signal. The Dual-Glo® Stop & Glo® Reagent quenched the firefly luciferase signal and sustained luminescence for the Renilla luciferase reaction. siRNA activity was determined by normalizing the Renilla (IGFALS) signal to the Firefly (control) signal within each well. The magnitude of siRNA activity was then assessed relative to cells that were transfected with the same vector but were not treated with siRNA or were treated with a non-targeting siRNA. All transfections were done in quadruplicates.

TABLE 12 Unmodified Sense and Antisense Strand Sequences of IGFALS dsRNAs Sense Position SEQ Antisense  Position in Duplex Oligo in NM_ ID Oligo NM_ SEQ ID Name Name Sense Sequence 004970.2 NO Name Antisense Sequence 004970.2 NO AD-76171 A-152158 CAAUUAAAGGCAAAGGCAAUA 2058-2078 1437 A-152159 UAUUGCCUUUGCCUUUAAUUGAU 2056-2078 1484 AD-76172 A-152160 ACACCUACAACAACAUCACCA 1808-1828 1438 A-152161 UGGUGAUGUUGUUGUAGGUGUAC 1806-1828 1485 AD-76173 A-152162 UACACCUACAACAACAUCACA 1807-1827 1439 A-152163 UGUGAUGUUGUUGUAGGUGUACG 1805-1827 1486 AD-76174 A-152164 CAUCAAGGCAAACGUGUUCGA  777-797 1440 A-152165 UCGAACACGUUUGCCUUGAUGGC  775-797 1487 AD-76175 A-152166 CACCUACAACAACAUCACCUA 1809-1829 1441 A-152167 UAGGUGAUGUUGUUGUAGGUGUA 1807-1829 1488 AD-76176 A-152168 CGUACACCUACAACAACAUCA 1805-1825 1442 A-152169 UGAUGUUGUUGUAGGUGUACGCG 1803-1825 1489 AD-76177 A-152170 GUACACCUACAACAACAUCAA 1806-1826 1443 A-152171 UUGAUGUUGUUGUAGGUGUACGC 1804-1826 1490 AD-76178 A-152172 GACUCUUCCUCAAGGACAACA 1322-1342 1444 A-152173 UGUUGUCCUUGAGGAAGAGUCGG 1320-1342 1491 AD-76179 A-152174 AGCCUUCCAGAACCUCUCCAA  357-377 1445 A-152175 UUGGAGAGGUUCUGGAAGGCUGC  355-377 1492 AD-76180 A-152176 UCACGCUAGACCACAACCAGA 1109-1129 1446 A-152177 UCUGGUUGUGGUCUAGCGUGAGC 1107-1129 1493 AD-76181 A-152178 UGGACGUCUCGCACAACCGCA 1541-1561 1447 A-152179 UGCGGUUGUGCGAGACGUCCAGC 1539-1561 1494 AD-76182 A-152180 ACCUGGACCGCAACCUCAUCA  824-844 1448 A-152181 UGAUGAGGUUGCGGUCCAGGUAG  822-844 1495 AD-76183 A-152182 UGGACCUGUCCCACAACCGCA  893-913 1449 A-152183 UGCGGUUGUGGGACAGGUCCAGC  891-913 1496 AD-76184 A-152184 UGCGGCUGUCCCACAACGCCA  965-985 1450 A-152185 UGGCGUUGUGGGACAGCCGCAGC  963-985 1497 AD-76185 A-152186 CGCUCGGCCUCAGCAACAACA  530-550 1451 A-152187 UGUUGUUGCUGAGGCCGAGCGAG  528-550 1498 AD-76186 A-152188 CCUCAGGAACAACUCACUGCA 1617-1637 1452 A-152189 UGCAGUGAGUUGUUCCUGAGGCU 1615-1637 1499 AD-76187 A-152190 CUGCGGACCUUCACGCCGCAA 1633-1653 1453 A-152191 UUGCGGCGUGAAGGUCCGCAGUG 1631-1653 1500 AD-76188 A-152192 CCUCUCUGGGAACUGUCUCCA 1185-1205 1454 A-152193 UGGAGACAGUUCCCAGAGAGGUU 1183-1205 1501 AD-76189 A-152194 GCUCUCCCGCAACCGCCUGGA 1473-1493 1455 A-152195 UCCAGGCGGUUGCGGGAGAGCAG 1471-1493 1502 AD-76190 A-152196 CGUCUCGCACAACCGCCUGGA 1545-1565 1456 A-152197 UCCAGGCGGUUGUGCGAGACGUC 1543-1565 1503 AD-76191 A-152198 CACGCUAGACCACAACCAGCA 1110-1130 1457 A-152199 UGCUGGUUGUGGUCUAGCGUGAG 1108-1130 1504 AD-76192 A-152200 UGCUCUCCCGCAACCGCCUGA 1472-1492 1458 A-152201 UCAGGCGGUUGCGGGAGAGCAGC 1470-1492 1505 AD-76193 A-152202 ACCUCAGCCUCAGGAACAACA 1610-1630 1459 A-152203 UGUUGUUCCUGAGGCUGAGGUAG 1608-1630 1506 AD-76194 A-152204 CACCUUCAAGGACCUGCACUA 1005-1025 1460 A-152205 UAGUGCAGGUCCUUGAAGGUGCG 1003-1025 1507 AD-76195 A-152206 GGCCUCGUGGGCAUUGAGGAA 1342-1362 1461 A-152207 UUCCUCAAUGCCCACGAGGCCGU 1340-1362 1508 AD-76196 A-152208 ACCUUCAAGGACCUGCACUUA 1006-1026 1462 A-152209 UAAGUGCAGGUCCUUGAAGGUGC 1004-1026 1509 AD-76197 A-152210 GGCCUUCUGGCUGGACGUCUA 1530-1550 1463 A-152211 UAGACGUCCAGCCAGAAGGCCCG 1528-1550 1510 AD-76198 A-152212 GGCAUUGAGGAGCAGAGCCUA 1351-1371 1464 A-152213 UAGGCUCUGCUCCUCAAUGCCCA 1349-1371 1511 AD-76199 A-152214 GCCUUCCAGAACCUCUCCAGA  358-378 1465 A-152215 UCUGGAGAGGUUCUGGAAGGCUG  356-378 1512 AD-76200 A-152216 GCGGUCAUGAACCUCUCUGGA 1174-1194 1466 A-152217 UCCAGAGAGGUUCAUGACCGCCA 1172-1194 1513 AD-76201 A-152218 AGGCUGGAGGACGGGCUCUUA  559-579 1467 A-152219 UAAGAGCCCGUCCUCCAGCCUGC  557-579 1514 AD-76202 A-152220 CUCUACCUGGACCGCAACCUA  820-840 1468 A-152221 UAGGUUGCGGUCCAGGUAGAGUU  818-840 1515 AD-76203 A-152222 GCGGAUGAGCUCAGCGUCUUA  238-258 1469 A-152223 UAAGACGCUGAGCUCAUCCGCGU  236-258 1516 AD-76204 A-152224 CCGUCUGAGCAGGCUGGAGGA  549-569 1470 A-152225 UCCUCCAGCCUGCUCAGACGGUU  547-569 1517 AD-76205 A-152226 GGUCAUGAACCUCUCUGGGAA 1176-1196 1471 A-152227 UUCCCAGAGAGGUUCAUGACCGC 1174-1196 1518 AD-76206 A-152228 CUGCAGCUGGGCCACAACCGA 1036-1056 1472 A-152229 UCGGUUGUGGCCCAGCUGCAGCU 1034-1056 1519 AD-76207 A-152230 CGGGAGCUGGACCUGAGCAGA  742-762 1473 A-152231 UCUGCUCAGGUCCAGCUCCCGGA  740-762 1520 AD-76208 A-152232 CCGCCUGUGUCUGCAGCUACA  209-229 1474 A-152233 UGUAGCUGCAGACACAGGCGGCC  207-229 1521 AD-76209 A-152234 UCUUCUGCAGCUCCAGGAACA  254-274 1475 A-152235 UGUUCCUGGAGCUGCAGAAGACG  252-274 1522 AD-76210 A-152236 CUGGCUGAGCGCAGCUUUGAA 1066-1086 1476 A-152237 UUCAAAGCUGCGCUCAGCCAGCU 1064-1086 1523 AD-76211 A-152238 CUCGACCUGACCUCCAACCAA 1396-1416 1477 A-152239 UUGGUUGGAGGUCAGGUCGAGCU 1394-1416 1524 AD-76212 A-152240 CUCAACCUCGGCUGGAAUAGA  604-624 1478 A-152241 UCUAUUCCAGCCGAGGUUGAGGU  602-624 1525 AD-76213 A-152242 GCCUAGAGAACCUGUGCCACA  440-460 1479 A-152243 UGUGGCACAGGUUCUCUAGGCCC  438-460 1526 AD-76214 A-152244 CAGCUUGAGGUGCUCACGCUA 1096-1116 1480 A-152245 UAGCGUGAGCACCUCAAGCUGCC 1094-1116 1527 AD-76215 A-152246 CAGGUCCUCAGUGUCCUCAGA 1961-1981 1481 A-152247 UCUGAGGACACUGAGGACCUGUC 1959-1981 1528 AD-76216 A-152248 GCUGCGAUGGCUGGACCUGUA  882-902 1482 A-152249 UACAGGUCCAGCCAUCGCAGCGC  880-902 1529 AD-76217 A-152250 GGCAAGCUGGAGUACCUGCUA 1453-1473 1483 A-152251 UAGCAGGUACUCCAGCUUGCCCA 1451-1473 1530

TABLE 13 IGFALS in vitro 10 nM and 0.1 nM screen Position in Duplex 10 nM 10 nM 0.1 nM 0.1 nM NM_ Name AVG STD AVG STD 004970.2 AD-76171 29.9 0.1 55.8 5.4 2058-2078 AD-76172 42.7 2.3 101.9 7.8 1808-1828 AD-76173 32.8 3.1 81.2 7.1 1807-1827 AD-76174 45.3 10.5 86.2 9.7 777-797 AD-76175 42.8 7.6 97.3 8.7 1809-1829 AD-76176 69 5.8 93.9 11.3 1805-1825 AD-76177 53.6 11 99.2 15.8 1806-1826 AD-76178 72.6 4.9 92.6 6.9 1322-1342 AD-76179 80.1 8.2 113.3 8 357-377 AD-76180 132 13.9 124.2 16.8 1109-1129 AD-76181 67.1 5.9 99.1 0.3 1541-1561 AD-76182 110.4 17.9 102 5.1 824-844 AD-76183 69.8 9.9 100.3 6.9 893-913 AD-76184 62 6.3 99.4 7.1 965-985 AD-76185 119.5 33.1 92.8 4.4 530-550 AD-76186 52.4 4.6 94.7 2.7 1617-1637 AD-76187 116.7 6.4 117.7 9.4 1633-1653 AD-76188 60.4 5.8 106.2 4.8 1185-1205 AD-76189 89.9 2.1 102.9 8.4 1473-1493 AD-76190 83.5 3.5 104.5 6.5 1545-1565 AD-76191 80.9 3.3 96.4 7.2 1110-1130 AD-76192 93.9 3.9 103 5.4 1472-1492 AD-76193 99.3 3.5 95.1 10.2 1610-1630 AD-76194 192.1 4.9 118 12.1 1005-1025 AD-76195 86.1 3.3 106.7 8.4 1342-1362 AD-76196 184 23.4 114.8 6.7 1006-1026 AD-76197 55.3 4.4 111.4 14.2 1530-1550 AD-76198 66.7 3.6 97.1 13.1 1351-1371 AD-76199 54.5 2 91.8 4.2 358-378 AD-76200 63.9 10.1 88 10.6 1174-1194 AD-76201 150.6 4.6 113.6 18.3 559-579 AD-76202 64.7 1.4 95.7 13.7 820-840 AD-76203 41.3 1.7 93 2.1 238-258 AD-76204 67.5 6.9 101.3 3.6 549-569 AD-76205 73.1 9.5 87.6 7.8 1176-1196 AD-76206 86.4 3.7 107.7 17 1036-1056 AD-76207 131.4 22.4 99.7 10.5 742-762 AD-76208 46.5 12 103 13.2 209-229 AD-76209 43.8 5.2 98.1 13.9 254-274 AD-76210 41.6 7.3 94.4 6 1066-1086 AD-76211 152.3 9.4 125.1 3.4 1396-1416 AD-76212 59.2 12.7 82.3 23.4 604-624 AD-76213 71.1 4.9 94.9 11.6 440-460 AD-76214 109.8 5.9 102.8 8.4 1096-1116 AD-76215 70.2 7.5 103.2 25.4 1961-1981 AD-76216 62.8 9.5 107.7 15.1 882-902 AD-76217 68.8 1.7 94.5 2.4 1453-1473 Mock 117.3 16.3 106.9 9.5

TABLE 14 Modified Sense and Antisense Strand Sequences of IGFALS dsRNAs Sense SEQ Antisense  SEQ SEQ Duplex Oligo ID Oligo ID ID Name Name Sense Sequence NO Name Antisense Sequence NO mRNA target sequence NO AD-76171 A-152158 csasauuaAfaGfGfCfaaaggcaauaL96 1531 A-152159 VPusAfsuugCfcUfUfugccUfuUfaauugsasu 1578 AUCAAUUAAAGGCAAAGGCAAUC 1625 AD-76172 A-152160 ascsaccuAfcAfAfCfaacaucaccaL96 1532 A-152161 VPusGfsgugAfuGfUfuguuGfuAfggugusasc 1579 GUACACCUACAACAACAUCACCU 1626 AD-76173 A-152162 usascaccUfaCfAfAfcaacaucacaL96 1533 A-152163 VPusGfsugaUfgUfUfguugUfaGfguguascsg 1580 CGUACACCUACAACAACAUCACC 1627 AD-76174 A-152164 csasucaaGfgCfAfAfacguguucgaL96 1534 A-152165 VPusCfsgaaCfaCfGfuuugCfcUfugaugsgsc 1581 GCCAUCAAGGCAAACGUGUUCGU 1628 AD-76175 A-152166 csasccuaCfaAfCfAfacaucaccuaL96 1535 A-152167 VPusAfsgguGfaUfGfuuguUfgUfaggugsusa 1582 UACACCUACAACAACAUCACCUG 1629 AD-76176 A-152168 csgsuacaCfcUfAfCfaacaacaucaL96 1536 A-152169 VPusGfsaugUfuGfUfuguaGfgUfguacgscsg 1583 CGCGUACACCUACAACAACAUCA 1630 AD-76177 A-152170 gsusacacCfuAfCfAfacaacaucaaT96 1537 A-152171 VPusUfsgauGfuUfGfuuguAfgGfuguacsgsc 1584 GCGUACACCUACAACAACAUCAC 1631 AD-76178 A-152172 gsascucuUfcCfUfCfaaggacaacaL96 1538 A-152173 VPusGfsuugUfcCfUfugagGfaAfgagucsgsg 1585 CCGACUCUUCCUCAAGGACAACG 1632 AD-76179 A-152174 asgsccuuCfcAfGfAfaccucuccaaL96 1539 A-152175 VPusUfsggaGfaGfGfuucuGfgAfaggcusgsc 1586 GCAGCCUUCCAGAACCUCUCCAG 1633 AD-76180 A-152176 uscsacgcUfaGfAfCfcacaaccagaL96 1540 A-152177 VPusCfsuggUfuGfUfggucUfaGfcgugasgsc 1587 GCUCACGCUAGACCACAACCAGC 1634 AD-76181 A-152178 usgsgacgUfcUfCfGfcacaaccgcaL96 1541 A-152179 VPusGfscggUfuGfUfgcgaGfaCfguccasgsc 1588 GCUGGACGUCUCGCACAACCGCC 1635 AD-76182 A-152180 ascscuggAfcCfGfCfaaccucaucaL96 1542 A-152181 VPusGfsaugAfgGfUfugcgGfuCfcaggusasg 1589 CUACCUGGACCGCAACCUCAUCG 1636 AD-76183 A-152182 usgsgaccUfgUfCfCfcacaaccgcaL96 1543 A-152183 VPusGfscggUfuGfUfgggaCfaGfguccasgsc 1590 GCUGGACCUGUCCCACAACCGCG 1637 AD-76184 A-152184 usgscggcUfgUfCfCfcacaacgccaL96 1544 A-152185 VPusGfsgcgUfuGfUfgggaCfaGfccgcasgsc 1591 GCUGCGGCUGUCCCACAACGCCA 1638 AD-76185 A-152186 csgscucgGfcCfUfCfagcaacaacaL96 1545 A-152187 VPusGfsuugUfuGfCfugagGfcCfgagcgsasg 1592 CUCGCUCGGCCUCAGCAACAACC 1639 AD-76186 A-152188 cscsucagGfaAfCfAfacucacugcaL96 1546 A-152189 VPusGfscagUfgAfGfuuguUfcCfugaggscsu 1593 AGCCUCAGGAACAACUCACUGCG 1640 AD-76187 A-152190 csusgcggAfcCfUfUfcacgccgcaaL96 1547 A-152191 VPusUfsgcgGfcGfUfgaagGfuCfcgcagsusg 1594 CACUGCGGACCUUCACGCCGCAG 1641 AD-76188 A-152192 cscsucucUfgGfGfAfacugucuccaL96 1548 A-152193 VPusGfsgagAfcAfGfuuccCfaGfagaggsusu 1595 AACCUCUCUGGGAACUGUCUCCG 1642 AD-76189 A-152194 gscsucucCfcGfCfAfaccgccuggaL96 1549 A-152195 VPusCfscagGfcGfGfuugcGfgGfagagcsasg 1596 CUGCUCUCCCGCAACCGCCUGGC 1643 AD-76190 A-152196 csgsucucGfcAfCfAfaccgccuggaL96 1550 A-152197 VPusCfscagGfcGfGfuuguGfcGfagacgsusc 1597 GACGUCUCGCACAACCGCCUGGA 1644 AD-76191 A-152198 csascgcuAfgAfCfCfacaaccagcaL96 1551 A-152199 VPusGfscugGfuUfGfugguCfuAfgcgugsasg 1598 CUCACGCUAGACCACAACCAGCU 1645 AD-76192 A-152200 usgscucuCfcCfGfCfaaccgccugaL96 1552 A-152201 VPusCfsaggCfgGfUfugcgGfgAfgagcasgsc 1599 GCUGCUCUCCCGCAACCGCCUGG 1646 AD-76193 A-152202 ascscucaGfcCfUfCfaggaacaacaL96 1553 A-152203 VPusGfsuugUfuCfCfugagGfcUfgaggusasg 1600 CUACCUCAGCCUCAGGAACAACU 1647 AD-76194 A-152204 csasccuuCfaAfGfGfaccugcacuaL96 1554 A-152205 VPusAfsgugCfaGfGfuccuUfgAfaggugscsg 1601 CGCACCUUCAAGGACCUGCACUU 1648 AD-76195 A-152206 gsgsccucGfuGfGfGfcauugaggaaT96 1555 A-152207 VPusUfsccuCfaAfUfgcccAfcGfaggccsgsu 1602 ACGGCCUCGUGGGCAUUGAGGAG 1649 AD-76196 A-152208 ascscuucAfaGfGfAfccugcacuuaL96 1556 A-152209 VPusAfsaguGfcAfGfguccUfuGfaaggusgsc 1603 GCACCUUCAAGGACCUGCACUUC 1650 AD-76197 A-152210 gsgsccuuCfuGfGfCfuggacgucuaL96 1557 A-152211 VPusAfsgacGfuCfCfagccAfgAfaggccscsg 1604 CGGGCCUUCUGGCUGGACGUCUC 1651 AD-76198 A-152212 gsgscauuGfaGfGfAfgcagagccuaL96 1558 A-152213 VPusAfsggcUfcUfGfcuccUfcAfaugccscsa 1605 UGGGCAUUGAGGAGCAGAGCCUG 1652 AD-76199 A-152214 gscscuucCfaGfAfAfccucuccagaL96 1559 A-152215 VPusCfsuggAfgAfGfguucUfgGfaaggcsusg 1606 CAGCCUUCCAGAACCUCUCCAGC 1653 AD-76200 A-152216 gscsggucAfuGfAfAfccucucuggaL96 1560 A-152217 VPusCfscagAfgAfGfguucAfuGfaccgcscsa 1607 UGGCGGUCAUGAACCUCUCUGGG 1654 AD-76201 A-152218 asgsgcugGfaGfGfAfcgggcucuuaL96 1561 A-152219 VPusAfsagaGfcCfCfguccUfcCfagccusgsc 1608 GCAGGCUGGAGGACGGGCUCUUC 1655 AD-76202 A-152220 csuscuacCfuGfGfAfccgcaaccuaL96 1562 A-152221 VPusAfsgguUfgCfGfguccAfgGfuagagsusu 1609 AACUCUACCUGGACCGCAACCUC 1656 AD-76203 A-152222 gscsggauGfaGfCfUfcagcgucuuaL96 1563 A-152223 VPusAfsagaCfgCfUfgagcUfcAfuccgcsgsu 1610 ACGCGGAUGAGCUCAGCGUCUUC 1657 AD-76204 A-152224 cscsgucuGfaGfCfAfggcuggaggaL96 1564 A-152225 VPusCfscucCfaGfCfcugcUfcAfgacggsusu 1611 AACCGUCUGAGCAGGCUGGAGGA 1658 AD-76205 A-152226 gsgsucauGfaAfCfCfucucugggaaL96 1565 A-152227 VPusUfscccAfgAfGfagguUfcAfugaccsgsc 1612 GCGGUCAUGAACCUCUCUGGGAA 1659 AD-76206 A-152228 csusgcagCfuGfGfGfccacaaccgaL96 1566 A-152229 VPusCfsgguUfgUfGfgcccAfgCfugcagscsu 1613 AGCUGCAGCUGGGCCACAACCGC 1660 AD-76207 A-152230 csgsggagCfuGfGfAfccugagcagaL96 1567 A-152231 VPusCfsugcUfcAfGfguccAfgCfucccgsgsa 1614 UCCGGGAGCUGGACCUGAGCAGG 1661 AD-76208 A-152232 cscsgccuGfuGfUfCfugcagcuacaL96 1568 A-152233 VPusGfsuagCfuGfCfagacAfcAfggcggscsc 1615 GGCCGCCUGUGUCUGCAGCUACG 1662 AD-76209 A-152234 uscsuucuGfcAfGfCfuccaggaacaL96 1569 A-152235 VPusGfsuucCfuGfGfagcuGfcAfgaagascsg 1616 CGUCUUCUGCAGCUCCAGGAACC 1663 AD-76210 A-152236 csusggcuGfaGfCfGfcagcuuugaaT96 1570 A-152237 VPusUfscaaAfgCfUfgcgcUfcAfgccagscsu 1617 AGCUGGCUGAGCGCAGCUUUGAG 1664 AD-76211 A-152238 csuscgacCfuGfAfCfcuccaaccaaT96 1571 A-152239 VPusUfsgguUfgGfAfggucAfgGfucgagscsu 1618 AGCUCGACCUGACCUCCAACCAG 1665 AD-76212 A-152240 csuscaacCfuCfGfGfcuggaauagaL96 1572 A-152241 VPusCfsuauUfcCfAfgccgAfgGfuugagsgsu 1619 ACCUCAACCUCGGCUGGAAUAGC 1666 AD-76213 A-152242 gscscuagAfgAfAfCfcugugccacaL96 1573 A-152243 VPusGfsuggCfaCfAfgguuCfuCfuaggcscsc 1620 GGGCCUAGAGAACCUGUGCCACC 1667 AD-76214 A-152244 csasgcuuGfaGfGfUfgcucacgcuaL96 1574 A-152245 VPusAfsgcgUfgAfGfcaccUfcAfagcugscsc 1621 GGCAGCUUGAGGUGCUCACGCUA 1668 AD-76215 A-152246 csasggucCfuCfAfGfuguccucagaL96 1575 A-152247 VPusCfsugaGfgAfCfacugAfgGfaccugsusc 1622 GACAGGUCCUCAGUGUCCUCAGG 1669 AD-76216 A-152248 gscsugcgAfuGfGfCfuggaccuguaL96 1576 A-152249 VPusAfscagGfuCfCfagccAfuCfgcagcsgsc 1623 GCGCUGCGAUGGCUGGACCUGUC 1670 AD-76217 A-152250 gsgscaagCfuGfGfAfguaccugcuaL96 1577 A-152251 VPusAfsgcaGfgUfAfcuccAfgCfuugccscsa 1624 UGGGCAAGCUGGAGUACCUGCUG 1671

Example 10—IGF-1 Transcripts, siRNA Design, and siRNA Screening Bioinformatics

A set of siRNAs targeting the human IGF1, (“insulin like growth factor 1”, NCBI refseqID: NM_000618; NCBI GeneID: 3479 (SEQ ID NO: 13), as well as toxicology-species IGF1 orthologs (cynomolgus monkey: XM_005572039) were designed using custom R and Python scripts. The human NM_000618 REFSEQ mRNA, version 3, has a length 7321 of bases.

The rationale and method for the set of siRNA designs is as follows: the predicted efficacy for every potential 19mer siRNA from position 10 through the end was determined with a linear model derived the direct measure of mRNA knockdown from more than 20,000 distinct siRNA designs targeting a large number of vertebrate genes. Subsets of the IGF1 siRNAs were designed with perfect or near-perfect matches between human and cynomolgus monkey. For each strand of the siRNA, a custom Python script was used in a brute force search to measure the number and positions of mismatches between the siRNA and all potential alignments in the target species transcriptome. Extra weight was given to mismatches in the seed region, defined here as positions 2-9 of the antisense oligonucleotide, as well the cleavage site of the siRNA, defined here as positions 10-11 of the antisense oligonucleotide. The relative weight of the mismatches was 2.8; 1.2:1 for seed mismatches, cleavage site, and other positions up through antisense position 19. Mismatches in the first position were ignored. A specificity score was calculated for each strand by summing the value of each weighted mismatch. Preference was given to siRNAs whose antisense score in human was >=2.2 and predicted efficacy was >=50% knockdown of the transcript.

In Vitro Dual-Glo® Screening

Cell Culture and Transfections

Cos7 cells (ATCC, Manassas, Va.) were grown to near confluence at 37° C. in an atmosphere of 5% CO₂ in DMEM (ATCC) supplemented with 10% FBS, before being released from the plate by trypsinization. Three human IGF-1 Dual-Glo® Luciferase constructs were generated using the psiCHECK2 vector. Construct one contained sequence based on NM_001111285 (SEQ ID NO: 1672), while constructs two and three contained sequence based on NM_000618 (SEQ ID NOs: 1673 and 1674). Dual-luciferase plasmids were co-transfected with siRNA into 5000 cells using Lipofectamine RNAiMax (Invitrogen, Carlsbad Calif. cat #13778-150). For each well of a 384 well plate, 0.1 μl of Lipofectamine was added to 5 ng of plasmid vector and siRNA in 15 μl of Opti-MEM and allowed to complex at room temperature for 15 minutes. The mixture was then added to the cells resuspended in 35 ul of fresh complete media. Cells were incubated for 48 hours before luciferase was measured. Screen was performed at 10 nM and 0.1 nM final duplex concentration.

Dual-Glo® Luciferase Assay

48 hours after the siRNAs were transfected, Firefly (transfection control) and Renilla (fused to IGF1 target sequence in 3′ UTR) luciferase were measured. First, media was removed from cells. Then Firefly luciferase activity was measured by adding 20 ul of Dual-Glo® Luciferase Reagent mixed with 20 ul of complete media to each well. The mixture was incubated at room temperature for 30 minutes before luminescense (500 nm) was measured on a Spectramax (Molecular Devices) to detect the Firefly luciferase signal. Renilla luciferase activity was measured by adding 20 ul of room temperature of Dual-Glo® Stop & Glo® Reagent to each well and the plates were incubated for 20 minutes before luminescence was again measured to determine the Renilla luciferase signal. The Dual-Glo® Stop & Glo® Reagent quenched the firefly luciferase signal and sustained luminescence for the Renilla luciferase reaction. siRNA activity was determined by normalizing the Renilla (IGF1) signal to the Firefly (control) signal within each well. The magnitude of siRNA activity was then assessed relative to cells that were transfected with the same vector but were not treated with siRNA or were treated with a non-targeting siRNA. All transfections were done in quadruplicates.

TABLE 15 Unmodified Sense and Antisense Strand Sequences of IGF-1dsRNAs Sense Position SEQ Antisense  Position in Duplex Oligo in NM_ ID Oligo NM_ SEQ ID Name Name Sense Sequence 000618.3 NO Name Antisense Sequence 000618.3 NO AD-75740 A-151647 AAUGUAACUAAUUGAAUCAUA 7181-7201 1675 A-151648 UAUGAUUCAAUUAGUUACAUUUU 7179-7201 1768 AD-75741 A-151649 UAAGAAAGUACUUGACUAAAA 4362-4382 1676 A-151650 UUUUAGUCAAGUACUUUCUUAAA 4360-4382 1769 AD-75747 A-151661 CUUUAUAAUUCUUGAAUGAGA 3765-3785 1677 A-151662 UCUCAUUCAAGAAUUAUAAAGCA 3763-3785 1770 AD-75748 A-151663 AGAUAGAAAUGUAUGUUUGAA 5223-5243 1678 A-151664 UUCAAACAUACAUUUCUAUCUAG 5221-5243 1771 AD-75749 A-151665 UUCCACAAUCCUUAGAAUCUA 6512-6532 1679 A-151666 UAGAUUCUAAGGAUUGUGGAAGG 6510-6532 1772 AD-75750 A-151667 UAUCAAAACCUUUCAAAUAUA 6831-6851 1680 A-151668 UAUAUUUGAAAGGUUUUGAUAUU 6829-6851 1773 AD-75751 A-151669 ACAAGUAAACAUUCCAACAUA  824-844 1681 A-151670 UAUGUUGGAAUGUUUACUUGUGU  822-844 1774 AD-75755 A-151677 ACAUAGAAAGUUUCUUCAACA 1410-1430 1682 A-151678 UGUUGAAGAAACUUUCUAUGUUU 1408-1430 1775 AD-75757 A-151681 CAGUCAACAAGUAUUUUAACA 3122-3142 1683 A-151682 UGUUAAAAUACUUGUUGACUGAA 3120-3142 1776 AD-75759 A-151685 CUCAAGCUGUCUACUUACAUA 6769-6789 1684 A-151686 UAUGUAAGUAGACAGCUUGAGGU 6767-6789 1777 AD-75760 A-151687 GAAUUGUUUCCUUAUUUGCAA 1085-1105 1685 A-151688 UUGCAAAUAAGGAAACAAUUCAU 1083-1105 1778 AD-75761 A-151689 AUCUGUCUUAGUUGAAAAGCA 7071-7091 1686 A-151690 UGCUUUUCAACUAAGACAGAUGU 7069-7091 1779 AD-75765 A-151697 AAUUAGCAAUAUAUUAUCCAA 4632-4652 1687 A-151698 UUGGAUAAUAUAUUGCUAAUUUU 4630-4652 1780 AD-75766 A-151699 AAUUGAAUCAUUAUCUUACAA 7190-7210 1688 A-151700 UUGUAAGAUAAUGAUUCAAUUAG 7188-7210 1781 AD-75769 A-151705 UUUAUCAAUAAUGUUCUAUAA  908-928 1689 A-151706 UUAUAGAACAUUAUUGAUAAAAG  906-928 1782 AD-75772 A-151711 GUAAAAGAAACUAUACAUCAA 3213-3233 1690 A-151712 UUGAUGUAUAGUUUCUUUUACAU 3211-3233 1783 AD-75774 A-151715 AAUGAGGAAUAAUAAGUUAAA 5173-5193 1691 A-151716 UUUAACUUAUUAUUCCUCAUUCU 5171-5193 1784 AD-75776 A-151719 CUCUGUGAAUGUGUUUUAUCA 2737-2757 1692 A-151720 UGAUAAAACACAUUCACAGAGAG 2735-2757 1785 AD-75778 A-151723 GUUCCUUCAAAUGAUGAGUUA 1438-1458 1693 A-151724 UAACUCAUCAUUUGAAGGAACUC 1436-1458 1786 AD-75779 A-151725 CAGGAUAAAGAUAUCAAUUUA 5722-5742 1694 A-151726 UAAAUUGAUAUCUUUAUCCUGUA 5720-5742 1787 AD-75787 A-151741 CAUGUCCUCCUCGCAUCUCUA  297-317 1695 A-151742 UAGAGAUGCGAGGAGGACAUGGU  295-317 1788 AD-75787 A-151741 CAUGUCCUCCUCGCAUCUCUA  297-317 1696 A-151742 UAGAGAUGCGAGGAGGACAUGGU  295-317 1789 AD-75788 A-151743 UUCAACAAGCCCACAGGGUAA  436-456 1697 A-151744 UUACCCUGUGGGCUUGUUGAAAU  434-456 1790 AD-75788 A-151743 UUCAACAAGCCCACAGGGUAA  436-456 1698 A-151744 UUACCCUGUGGGCUUGUUGAAAU  434-456 1791 AD-75789 A-151745 UCCUCGCAUCUCUUCUACCUA  304-324 1699 A-151746 UAGGUAGAAGAGAUGCGAGGAGG  302-324 1792 AD-75789 A-151745 UCCUCGCAUCUCUUCUACCUA  304-324 1700 A-151746 UAGGUAGAAGAGAUGCGAGGAGG  302-324 1793 AD-75790 A-151747 GGGGCUUUUAUUUCAACAAGA  425-445 1701 A-151748 UCUUGUUGAAAUAAAAGCCCCUG  423-445 1794 AD-75790 A-151747 GGGGCUUUUAUUUCAACAAGA  425-445 1702 A-151748 UCUUGUUGAAAUAAAAGCCCCUG  423-445 1795 AD-75791 A-151749 GAUGCUCUUCAGUUCGUGUGA  397-417 1703 A-151750 UCACACGAACUGAAGAGCAUCCA  395-417 1796 AD-75791 A-151749 GAUGCUCUUCAGUUCGUGUGA  397-417 1704 A-151750 UCACACGAACUGAAGAGCAUCCA  395-417 1797 AD-75792 A-151751 UGGAUGCUCUUCAGUUCGUGA  395-415 1705 A-151752 UCACGAACUGAAGAGCAUCCACC  393-415 1798 AD-75792 A-151751 UGGAUGCUCUUCAGUUCGUGA  395-415 1706 A-151752 UCACGAACUGAAGAGCAUCCACC  393-415 1799 AD-75793 A-151753 GGUGGAUGCUCUUCAGUUCGA  393-413 1707 A-151754 UCGAACUGAAGAGCAUCCACCAG  391-413 1800 AD-75793 A-151753 GGUGGAUGCUCUUCAGUUCGA  393-413 1708 A-151754 UCGAACUGAAGAGCAUCCACCAG  391-413 1801 AD-75794 A-151755 GCUGGUGGAUGCUCUUCAGUA  390-410 1709 A-151756 UACUGAAGAGCAUCCACCAGCUC  388-410 1802 AD-75794 A-151755 GCUGGUGGAUGCUCUUCAGUA  390-410 1710 A-151756 UACUGAAGAGCAUCCACCAGCUC  388-410 1803 AD-75795 A-151757 CUGGUGGAUGCUCUUCAGUUA  391-411 1711 A-151758 UAACUGAAGAGCAUCCACCAGCU  389-411 1804 AD-75795 A-151757 CUGGUGGAUGCUCUUCAGUUA  391-411 1712 A-151758 UAACUGAAGAGCAUCCACCAGCU  389-411 1805 AD-77120 A-154752 CCAAAAUGCACUGAUGUAAAA 6586-6606 1713 A-154753 UUUUACAUCAGUGCAUUUUGGGC 6584-6606 1806 AD-77121 A-154754 UCCAGUUGCACUAAAUUCCUA 1009-1029 1714 A-154755 UAGGAAUUUAGUGCAACUGGAUC 1007-1029 1807 AD-77122 A-154756 CAUUCUUCAACUAUCUUUGAA 6325-6345 1715 A-154757 UUCAAAGAUAGUUGAAGAAUGAG 6323-6345 1808 AD-77123 A-154758 ACAUUCCAACAUUGUCUUUAA  832-852 1716 A-154759 UUAAAGACAAUGUUGGAAUGUUU  830-852 1809 AD-77124 A-154760 GGCUUAGAAUAAAAGAUGUAA 4280-4300 1717 A-154761 UUACAUCUUUUAUUCUAAGCCUU 4278-4300 1810 AD-77125 A-154762 UUUCAAGAUAUUUGUAAAAGA 3789-3809 1718 A-154763 UCUUUUACAAAUAUCUUGAAAUU 3787-3809 1811 AD-77126 A-154764 AUAAGCAUAUUUUGAAAAUGA 7221-7241 1719 A-154765 UCAUUUUCAAAAUAUGCUUAUUA 7219-7241 1812 AD-77127 A-154766 GUUUUCAAUGCUAGUGUUUAA 5553-5573 1720 A-154767 UUAAACACUAGCAUUGAAAACAA 5551-5573 1813 AD-77128 A-154768 CAAUGAAAUACACAAGUAAAA  813-833 1721 A-154769 UUUUACUUGUGUAUUUCAUUGGG  811-833 1814 AD-77129 A-154770 CAAAAGUCCACUGAUGCAAAA 1764-1784 1722 A-154771 UUUUGCAUCAGUGGACUUUUGUG 1762-1784 1815 AD-77130 A-154772 AACAUAGAAAGUUUCUUCAAA 1409-1429 1723 A-154773 UUUGAAGAAACUUUCUAUGUUUA 1407-1429 1816 AD-77131 A-154774 AACCUAAUUAGUAACUUUCCA 1465-1485 1724 A-154775 UGGAAAGUUACUAAUUAGGUUGC 1463-1485 1817 AD-77132 A-154776 GACUUAUUUCCUUCACUUAAA 3442-3462 1725 A-154777 UUUAAGUGAAGGAAAUAAGUCAU 3440-3462 1818 AD-77133 A-154778 GGCUCUCAAACUUAGCAAAAA 4614-4634 1726 A-154779 UUUUUGCUAAGUUUGAGAGCCAA 4612-4634 1819 AD-77134 A-154780 ACAAAGAUAAGUGAAAAGAGA 2312-2332 1727 A-154781 UCUCUUUUCACUUAUCUUUGUAU 2310-2332 1820 AD-77135 A-154782 GUAGUGUACUGUUCACCAAAA 4525-4545 1728 A-154783 UUUUGGUGAACAGUACACUACUU 4523-4545 1821 AD-77136 A-154784 UCACCAACUCAUAGCAAAGUA 6663-6683 1729 A-154785 UACUUUGCUAUGAGUUGGUGAGU 6661-6683 1822 AD-77137 A-154786 AAAAUAACUCAUUAUACCAAA 3577-3597 1730 A-154787 UUUGGUAUAAUGAGUUAUUUUCA 3575-3597 1823 AD-77138 A-154788 AAGAAAGAAUCUUUCCAUUCA 5344-5364 1731 A-154789 UGAAUGGAAAGAUUCUUUCUUCU 5342-5364 1824 AD-77139 A-154790 AUUUCAAGAUAUUUGUAAAAA 3788-3808 1732 A-154791 UUUUUACAAAUAUCUUGAAAUUG 3786-3808 1825 AD-77140 A-154792 AGCUGAAACUCUAGAAUUAAA 5908-5928 1733 A-154793 UUUAAUUCUAGAGUUUCAGCUUG 5906-5928 1826 AD-77141 A-154794 AAAGAUAAAUCUGAUUUAUGA 5394-5414 1734 A-154795 UCAUAAAUCAGAUUUAUCUUUUG 5392-5414 1827 AD-77142 A-154796 UAUUAAAUUAAUUCUGAUUGA 7099-7119 1735 A-154797 UCAAUCAGAAUUAAUUUAAUAAA 7097-7119 1828 AD-77143 A-154798 AAGAAUGAGCUUUCAACUCAA 3825-3845 1736 A-154799 UUGAGUUGAAAGCUCAUUCUUAC 3823-3845 1829 AD-77144 A-154800 GUUUGAUAACAUUUAAAAGAA  780-800 1737 A-154801 UUCUUUUAAAUGUUAUCAAACUU  778-800 1830 AD-77145 A-154802 UGUUUUAAACAUAGAAAGUUA 1402-1422 1738 A-154803 UAACUUUCUAUGUUUAAAACAUA 1400-1422 1831 AD-77146 A-154804 UAAAUAACCCAUUCAUAGCAA 2110-2130 1739 A-154805 UUGCUAUGAAUGGGUUAUUUAUA 2108-2130 1832 AD-77147 A-154806 CACAUAGACUCUUUAAAACUA 5196-5216 1740 A-154807 UAGUUUUAAAGAGUCUAUGUGGG 5194-5216 1833 AD-77148 A-154808 AAGUCACAAAGUUAUCUUCUA 2568-2588 1741 A-154809 UAGAAGAUAACUUUGUGACUUUU 2566-2588 1834 AD-77149 A-154810 UAAAGAAAGAAUUUAUGAGAA 5011-5031 1742 A-154811 UUCUCAUAAAUUCUUUCUUUAUC 5009-5031 1835 AD-77150 A-154812 GAAAUGUAUGUUUGACUUGUA 5228-5248 1743 A-154813 UACAAGUCAAACAUACAUUUCUA 5226-5248 1836 AD-77151 A-154814 UUUAUAAGACCUUCCUGUUAA 5611-5631 1744 A-154815 UUAACAGGAAGGUCUUAUAAAAU 5609-5631 1837 AD-77152 A-154816 CUGUGAAUGUGUUUUAUCCAA 2739-2759 1745 A-154817 UUGGAUAAAACACAUUCACAGAG 2737-2759 1838 AD-77153 A-154818 UUAAUUCUGAUUGUAUUUGAA 7106-7126 1746 A-154819 UUCAAAUACAAUCAGAAUUAAUU 7104-7126 1839 AD-77154 A-154820 UGUCAUCUACCUACCUCAAAA 1982-2002 1747 A-154821 UUUUGAGGUAGGUAGAUGACAUA 1980-2002 1840 AD-77155 A-154822 CGUGAAAGCAAAACAAUAGGA 1634-1654 1748 A-154823 UCCUAUUGUUUUGCUUUCACGUA 1632-1654 1841 AD-77156 A-154824 AACAACAAUUCUAUAGAUAGA 4438-4458 1749 A-154825 UCUAUCUAUAGAAUUGUUGUUUU 4436-4458 1842 AD-77157 A-154826 UAAAGUAUUAUUUGAACUUUA 3920-3940 1750 A-154827 UAAAGUUCAAAUAAUACUUUAAU 3918-3940 1843 AD-77158 A-154828 AGAAAGAAUUUAUGAGAAAUA 5014-5034 1751 A-154829 UAUUUCUCAUAAAUUCUUUCUUU 5012-5034 1844 AD-77159 A-154830 AAAUGCACUGAUGUAAAGUAA 6589-6609 1752 A-154831 UUACUUUACAUCAGUGCAUUUUG 6587-6609 1845 AD-77160 A-154832 AUUAAUUCUGAUUGUAUUUGA 7105-7125 1753 A-154833 UCAAAUACAAUCAGAAUUAAUUU 7103-7125 1846 AD-77161 A-154834 CAAUAAUGUUCUAUAGAAAAA  913-933 1754 A-154835 UUUUUCUAUAGAACAUUAUUGAU  911-933 1847 AD-77162 A-154836 GAUUUUCAAUUUGAUUUUGAA 2269-2289 1755 A-154837 UUCAAAAUCAAAUUGAAAAUCAA 2267-2289 1848 AD-77163 A-154840 UUGAUUUUGAAUUCUGCAUUA 2279-2299 1756 A-154841 UAAUGCAGAAUUCAAAAUCAAAU 2277-2299 1849 AD-77164 A-154842 UUUCAAUUUGAUUUUGAAUUA 2272-2292 1757 A-154843 UAAUUCAAAAUCAAAUUGAAAAU 2270-2292 1850 AD-77165 A-154844 GUGUCUGUGUAUCAUGAAAAA 6798-6818 1758 A-154845 UUUUUCAUGAUACACAGACACAG 6796-6818 1851 AD-77166 A-154846 GGUUUUAUGAAUACAAAGAUA 2300-2320 1759 A-154847 UAUCUUUGUAUUCAUAAAACCAA 2298-2320 1852 AD-77167 A-154848 GCAGAUCAAGAUUUUCUCAUA 2837-2857 1760 A-154849 UAUGAGAAAAUCUUGAUCUGCAG 2835-2857 1853 AD-77168 A-154850 AACUAAUUGAAUCAUUAUCUA 7186-7206 1761 A-154851 UAGAUAAUGAUUCAAUUAGUUAC 7184-7206 1854 AD-77169 A-154852 UGGUUUAUGAAUUGUUUCCUA 1077-1097 1762 A-154853 UAGGAAACAAUUCAUAAACCACU 1075-1097 1855 AD-77170 A-154854 UAGUAUAAUGGUGCUAUUUUA 1574-1594 1763 A-154855 UAAAAUAGCACCAUUAUACUAAA 1572-1594 1856 AD-77171 A-154856 CUGUUUAAUAAGCAUAUUUUA 7214-7234 1764 A-154857 UAAAAUAUGCUUAUUAAACAGUA 7212-7234 1857 AD-77172 A-154858 AAAUAUCAAAACCUUUCAAAA 6828-6848 1765 A-154859 UUUUGAAAGGUUUUGAUAUUUUG 6826-6848 1858 AD-77173 A-154860 ACAGAUGUAAAAGAAACUAUA 3207-3227 1766 A-154861 UAUAGUUUCUUUUACAUCUGUCC 3205-3227 1859 AD-77174 A-154862 AACUUUGAGGCCAAUCAUUUA 1373-1393 1767 A-154863 UAAAUGAUUGGCCUCAAAGUUGC 1371-1393 1860

TABLE 16 IGF-1 in vitro 10 nM and 0.1 nM screen Duplex 10 nM 10 nM 0.1 nM 0.1 nM Position in Name AVG STD AVG STD NM_000618.3 AD-75740 2.9 1.1 20.9 1.6 7179-7201 AD-75741 9.2 1.5 55.6 9.3 4360-4382 AD-75747 50.3 6.8 67.7 8.8 3763-3785 AD-75748 8.4 1.1 36 10.1 5221-5243 AD-75749 7.8 1.3 65.9 3 6510-6532 AD-75750 4.4 1.3 48 9.3 6829-6851 AD-75751 5.7 1.4 51.7 9.4 822-844 AD-75755 29.4 7.2 60.9 3.8 1408-1430 AD-75757 32.4 5.7 62.3 7.9 3120-3142 AD-75759 9.8 4.3 86.5 11.1 6767-6789 AD-75760 32.2 7.8 56.2 7.1 1083-1105 AD-75761 3.5 1.3 53.3 4.3 7069-7091 AD-75765 7.6 1.9 56.5 8.6 4630-4652 AD-75766 3.3 2.4 35.4 7.4 7188-7210 AD-75769 12.4 3.1 33.6 3.9 906-928 AD-75772 36.1 5.2 79.9 19.3 3211-3233 AD-75774 14 1.2 51.8 7.8 5171-5193 AD-75776 41.1 11.6 84.2 10.3 2735-2757 AD-75778 42.3 7.4 56.4 6.6 1436-1458 AD-75779 8.7 1.6 53.7 9.1 5720-5742 AD-75787 59.5 5.5 96.5 6.2 295-317 AD-75787 57.5 12.9 99.5 10.4 295-317 AD-75788 38.5 5.2 82.2 9.3 434-456 AD-75788 28.1 2.1 88.3 4.4 434-456 AD-75789 58 11.5 81.6 3 302-324 AD-75789 65.1 16.2 93.6 5.6 302-324 AD-75790 54.7 5.9 90.1 1.7 423-445 AD-75790 64.1 6.1 92.6 4.3 423-445 AD-75791 17.4 5 78.8 3.9 395-417 AD-75791 19.5 2.1 87.5 7.1 395-417 AD-75792 12.7 1.5 63.7 6.2 393-415 AD-75792 13.9 2 77.8 12.9 393-415 AD-75793 23.6 4.1 78.5 4.7 391-413 AD-75793 27.8 4.1 90.9 11.6 391-413 AD-75794 40.6 3.1 89 12.3 388-410 AD-75794 46.7 4.4 91.1 9.6 388-410 AD-75795 32.2 8.2 75.2 6.3 389-411 AD-75795 27.3 5.3 77.7 7.3 389-411 AD-77120 17.7 1.8 83.5 3.7 6584-6606 AD-77121 28.3 3.4 69 5.9 1007-1029 AD-77122 19.3 6.4 80.4 10.5 6323-6345 AD-77123 21.2 4.1 46.9 8 830-852 AD-77124 13.9 1.5 35.3 5.8 4278-4300 AD-77125 44.5 10.3 52.4 6.9 3787-3809 AD-77126 4.5 3 23.6 3.9 7219-7241 AD-77127 8.3 1.5 43.1 4.9 5551-5573 AD-77128 13.6 4.1 46.6 5.3 811-833 AD-77129 68.5 7.6 99.9 8.2 1762-1784 AD-77130 32.1 5.8 41.4 3.4 1407-1429 AD-77131 36 8.5 53.8 7.2 1463-1485 AD-77132 44.3 8.2 71.7 5.1 3440-3462 AD-77133 21.4 6.4 88.9 5.8 4612-4634 AD-77134 37.9 2.4 82.4 8.7 2310-2332 AD-77135 19.6 5 91.8 17 4523-4545 AD-77136 32.3 8 89.5 3.5 6661-6683 AD-77137 36.8 3.3 67.7 7.7 3575-3597 AD-77138 2.6 1.9 71.3 6.3 5342-5364 AD-77139 45.6 2.5 70.4 8.2 3786-3808 AD-77140 17.1 3.2 65.4 6.1 5906-5928 AD-77141 56.3 17.6 112.7 8.2 5392-5414 AD-77142 18.1 3.7 65.7 8.4 7097-7119 AD-77143 45.7 10.1 93.6 11 3823-3845 AD-77144 5.5 2 43.5 7.6 778-800 AD-77145 59.6 11.1 43.8 5.1 1400-1422 AD-77146 27.7 5.8 64.8 7.7 2108-2130 AD-77147 6.6 5.4 75 10.2 5194-5216 AD-77148 37.6 6.7 65.9 8 2566-2588 AD-77149 5.5 0.7 35.8 5.2 5009-5031 AD-77150 1.1 2.3 48.2 5.1 5226-5248 AD-77151 9.1 2.3 69.2 5.9 5609-5631 AD-77152 52.6 5.9 93.7 6.5 2737-2759 AD-77153 10.4 1 37.8 6.1 7104-7126 AD-77154 57.5 14.5 101.7 11.5 1980-2002 AD-77155 63.3 8.5 53 3.9 1632-1654 AD-77156 14.8 3 47.9 8.9 4436-4458 AD-77158 1.3 1 44.3 0.9 5012-5034 AD-77159 20.5 4.9 92.3 17.2 6587-6609 AD-77160 10.8 5.2 39.3 4.9 7103-7125 AD-77161 26.1 1.8 47.3 6.8 911-933 AD-77162 51.6 5.4 93.6 3.1 2267-2289 AD-77163 41.2 4.4 70 3.1 2277-2299 AD-77164 58.5 5.8 95.5 10.9 2270-2292 AD-77165 11.1 2 61.7 7.1 6796-6818 AD-77166 40.3 5.6 74.2 1.7 2298-2320 AD-77167 55 9.2 84.6 18 2835-2857 AD-77168 7.9 0.6 28.4 3.6 7184-7206 AD-77169 36.1 7.5 51.6 5.6 1075-1097 AD-77170 44.4 8.8 66.7 3.6 1572-1594 AD-77171 13.1 1.6 50.1 9.3 7212-7234 AD-77172 38.8 5.4 103.7 12.7 6826-6848 AD-77173 42.4 2.8 79.8 15.2 3205-3227 AD-77174 48.9 3.5 83.3 6.5 1371-1393 Mock 100 6.1 100 5.6

TABLE 17 Modified Sense and Antisense Strand Sequences of IGF-1 dsRNAs Sense SEQ Antisense  SEQ SEQ Duplex Oligo ID Oligo ID ID Name Name Sense Sequence NO Name Antisense Sequence NO mRNA target sequence NO AD-75740 A-151647 asasuguaAfcUfAfAfuugaaucauaL96 1861 A-151648 VPusAfsugaUfuCfAfauuaGfuUfacauususu 1954 AAAAUGUAACUAAUUGAAUCAUU 2047 AD-75741 A-151649 usasagaaAfgUfAfCfuugacuaaaaL96 1862 A-151650 VPusUfsuuaGfuCfAfaguaCfuUfucuuasasa 1955 UUUAAGAAAGUACUUGACUAAAA 2048 AD-75747 A-151661 csusuuauAfaUfUfCfuugaaugagaL96 1863 A-151662 VPusCfsucaUfuCfAfagaaUfuAfuaaagscsa 1956 UGCUUUAUAAUUCUUGAAUGAGG 2049 AD-75748 A-151663 asgsauagAfaAfUfGfuauguuugaaL96 1864 A-151664 VPusUfscaaAfcAfUfacauUfuCfuaucusasg 1957 CUAGAUAGAAAUGUAUGUUUGAC 2050 AD-75749 A-151665 ususccacAfaUfCfCfuuagaaucuaL96 1865 A-151666 VPusAfsgauUfcUfAfaggaUfuGfuggaasgsg 1958 CCUUCCACAAUCCUUAGAAUCUG 2051 AD-75750 A-151667 usasucaaAfaCfCfUfuucaaauauaL96 1866 A-151668 VPusAfsuauUfuGfAfaaggUfuUfugauasusu 1959 AAUAUCAAAACCUUUCAAAUAUC 2052 AD-75751 A-151669 ascsaaguAfaAfCfAfuuccaacauaL96 1867 A-151670 VPusAfsuguUfgGfAfauguUfuAfcuugusgsu 1960 ACACAAGUAAACAUUCCAACAUU 2053 AD-75755 A-151677 ascsauagAfaAfGfUfuucuucaacaL96 1868 A-151678 VPusGfsuugAfaGfAfaacuUfuCfuaugususu 1961 AAACAUAGAAAGUUUCUUCAACU 2054 AD-75757 A-151681 csasgucaAfcAfAfGfuauuuuaacaL96 1869 A-151682 VPusGfsuuaAfaAfUfacuuGfuUfgacugsasa 1962 UUCAGUCAACAAGUAUUUUAACU 2055 AD-75759 A-151685 csuscaagCfuGfUfCfuacuuacauaL96 1870 A-151686 VPusAfsuguAfaGfUfagacAfgCfuugagsgsu 1963 ACCUCAAGCUGUCUACUUACAUC 2056 AD-75760 A-151687 gsasauugUfuUTCfCfuuauuugcaaL96 1871 A-151688 VPusUfsgcaAfaUfAfaggaAfaCfaauucsasu 1964 AUGAAUUGUUUCCUUAUUUGCAC 2057 AD-75761 A-151689 asuscuguCfuUfAfGfuugaaaagcaL96 1872 A-151690 VPusGfscuuUfuCfAfacuaAfgAfcagausgsu 1965 ACAUCUGUCUUAGUUGAAAAGCA 2058 AD-75765 A-151697 asasuuagCfaAfUfAfuauuauccaaL96 1873 A-151698 VPusUfsggaUfaAfUfauauUfgCfuaauususu 1966 AAAAUUAGCAAUAUAUUAUCCAA 2059 AD-75766 A-151699 asasuugaAfuCfAfUfuaucuuacaaL96 1874 A-151700 VPusUfsguaAfgAfUfaaugAfuUfcaauusasg 1967 CUAAUUGAAUCAUUAUCUUACAU 2060 AD-75769 A-151705 ususuaucAfaUfAfAfuguucuauaaL96 1875 A-151706 VPusUfsauaGfaAfCfauuaUfuGfauaaasasg 1968 CUUUUAUCAAUAAUGUUCUAUAG 2061 AD-75772 A-151711 gsusaaaaGfaAfAfCfuauacaucaaL96 1876 A-151712 VPusUfsgauGfuAfUfaguuUfcUfuuuacsasu 1969 AUGUAAAAGAAACUAUACAUCAU 2062 AD-75774 A-151715 asasugagGfaAfUfAfauaaguuaaaL96 1877 A-151716 VPusUfsuaaCfuUfAfuuauUfcCfucauuscsu 1970 AGAAUGAGGAAUAAUAAGUUAAA 2063 AD-75776 A-151719 csuscuguGfaAfUfGfuguuuuaucaL96 1878 A-151720 VPusGfsauaAfaAfCfacauUfcAfcagagsasg 1971 CUCUCUGUGAAUGUGUUUUAUCC 2064 AD-75778 A-151723 gsusuccuUfcAfAfAfugaugaguuaL96 1879 A-151724 VPusAfsacuCfaUfCfauuuGfaAfggaacsusc 1972 GAGUUCCUUCAAAUGAUGAGUUA 2065 AD-75779 A-151725 csasggauAfaAfGfAfuaucaauuuaL96 1880 A-151726 VPusAfsaauUfgAfUfaucuUfuAfuccugsusa 1973 UACAGGAUAAAGAUAUCAAUUUA 2066 AD-75787 A-151741 csasugucCfuCfCfUfcgcaucucuaL96 1881 A-151742 VPusAfsgagAfuGfCfgaggAfgGfacaugsgsu 1974 ACCAUGUCCUCCUCGCAUCUCUU 2067 AD-75787 A-151741 csasugucCfuCfCfUfcgcaucucuaL96 1882 A-151742 VPusAfsgagAfuGfCfgaggAfgGfacaugsgsu 1975 ACCAUGUCCUCCUCGCAUCUCUU 2068 AD-75788 A-151743 ususcaacAfaGfCfCfcacaggguaaL96 1883 A-151744 VPusUfsaccCfuGfUfgggcUfuGfuugaasasu 1976 AUUUCAACAAGCCCACAGGGUAU 2069 AD-75788 A-151743 ususcaacAfaGfCfCfcacaggguaaL96 1884 A-151744 VPusUfsaccCfuGfUfgggcUfuGfuugaasasu 1977 AUUUCAACAAGCCCACAGGGUAU 2070 AD-75789 A-151745 uscscucgCfaUfCfUfcuucuaccuaL96 1885 A-151746 VPusAfsgguAfgAfAfgagaUfgCfgaggasgsg 1978 CCUCCUCGCAUCUCUUCUACCUG 2071 AD-75789 A-151745 uscscucgCfaUfCfUfcuucuaccuaL96 1886 A-151746 VPusAfsgguAfgAfAfgagaUfgCfgaggasgsg 1979 CCUCCUCGCAUCUCUUCUACCUG 2072 AD-75790 A-151747 gsgsggcuUfuUfAfUfuucaacaagaL96 1887 A-151748 VPusCfsuugUfuGfAfaauaAfaAfgccccsusg 1980 CAGGGGCUUUUAUUUCAACAAGC 2073 AD-75790 A-151747 gsgsggcuUfuUfAfUfuucaacaagaL96 1888 A-151748 VPusCfsuugUfuGfAfaauaAfaAfgccccsusg 1981 CAGGGGCUUUUAUUUCAACAAGC 2074 AD-75791 A-151749 gsasugcuCfuUfCfAfguucgugugaL96 1889 A-151750 VPusCfsacaCfgAfAfcugaAfgAfgcaucscsa 1982 UGGAUGCUCUUCAGUUCGUGUGU 2075 AD-75791 A-151749 gsasugcuCfuUfCfAfguucgugugaL96 1890 A-151750 VPusCfsacaCfgAfAfcugaAfgAfgcaucscsa 1983 UGGAUGCUCUUCAGUUCGUGUGU 2076 AD-75792 A-151751 usgsgaugCfuCfUfUfcaguucgugaL96 1891 A-151752 VPusCfsacgAfaCfUfgaagAfgCfauccascsc 1984 GGUGGAUGCUCUUCAGUUCGUGU 2077 AD-75792 A-151751 usgsgaugCfuCfUfUfcaguucgugaL96 1892 A-151752 VPusCfsacgAfaCfUfgaagAfgCfauccascsc 1985 GGUGGAUGCUCUUCAGUUCGUGU 2078 AD-75793 A-151753 gsgsuggaUfgCfUfCfuucaguucgaL96 1893 A-151754 VPusCfsgaaCfuGfAfagagCfaUfccaccsasg 1986 CUGGUGGAUGCUCUUCAGUUCGU 2079 AD-75793 A-151753 gsgsuggaUfgCfUfCfuucaguucgaL96 1894 A-151754 VPusCfsgaaCfuGfAfagagCfaUfccaccsasg 1987 CUGGUGGAUGCUCUUCAGUUCGU 2080 AD-75794 A-151755 gscsugguGfgAfUfGfcucuucaguaL96 1895 A-151756 VPusAfscugAfaGfAfgcauCfcAfccagcsusc 1988 GAGCUGGUGGAUGCUCUUCAGUU 2081 AD-75794 A-151755 gscsugguGfgAfUfGfcucuucaguaL96 1896 A-151756 VPusAfscugAfaGfAfgcauCfcAfccagcsusc 1989 GAGCUGGUGGAUGCUCUUCAGUU 2082 AD-75795 A-151757 csusggugGfaUfGfCfucuucaguuaL96 1897 A-151758 VPusAfsacuGfaAfGfagcaUfcCfaccagscsu 1990 AGCUGGUGGAUGCUCUUCAGUUC 2083 AD-75795 A-151757 csusggugGfaUfGfCfucuucaguuaL96 1898 A-151758 VPusAfsacuGfaAfGfagcaUfcCfaccagscsu 1991 AGCUGGUGGAUGCUCUUCAGUUC 2084 AD-77120 A-154752 cscsaaaaUfgCfAfCfugauguaaaaL96 1899 A-154753 VPusUfsuuaCfaUfCfagugCfaUfuuuggsgsc 1992 GCCCAAAAUGCACUGAUGUAAAG 2085 AD-77121 A-154754 uscscaguUfgCfAfCfuaaauuccuaL96 1900 A-154755 VPusAfsggaAfuUfUfagugCfaAfcuggasusc 1993 GAUCCAGUUGCACUAAAUUCCUC 2086 AD-77122 A-154756 csasuucuUfcAfAfCfuaucuuugaaL96 1901 A-154757 VPusUfscaaAfgAfUfaguuGfaAfgaaugsasg 1994 CUCAUUCUUCAACUAUCUUUGAU 2087 AD-77123 A-154758 ascsauucCfaAfCfAfuugucuuuaaL96 1902 A-154759 VPusUfsaaaGfaCfAfauguUfgGfaaugususu 1995 AAACAUUCCAACAUUGUCUUUAG 2088 AD-77124 A-154760 gsgscuuaGfaAfUfAfaaagauguaaL96 1903 A-154761 VPusUfsacaUfcUfUfuuauUfcUfaagccsusu 1996 AAGGCUUAGAAUAAAAGAUGUAG 2089 AD-77125 A-154762 ususucaaGfaUfAfUfuuguaaaagaL96 1904 A-154763 VPusCfsuuuUfaCfAfaauaUfcUfugaaasusu 1997 AAUUUCAAGAUAUUUGUAAAAGA 2090 AD-77126 A-154764 asusaagcAfuAfUfUfuugaaaaugaL96 1905 A-154765 VPusCfsauuUfuCfAfaaauAfuGfcuuaususa 1998 UAAUAAGCAUAUUUUGAAAAUGU 2091 AD-77127 A-154766 gsusuuucAfaUfGfCfuaguguuuaaL96 1906 A-154767 VPusUfsaaaCfaCfUfagcaUfuGfaaaacsasa 1999 UUGUUUUCAAUGCUAGUGUUUAA 2092 AD-77128 A-154768 csasaugaAfaUfAfCfacaaguaaaaL96 1907 A-154769 VPusUfsuuaCfuUfGfuguaUfuUfcauugsgsg 2000 CCCAAUGAAAUACACAAGUAAAC 2093 AD-77129 A-154770 csasaaagUfcCfAfCfugaugcaaaaL96 1908 A-154771 VPusUfsuugCfaUfCfagugGfaCfuuuugsusg 2001 CACAAAAGUCCACUGAUGCAAAU 2094 AD-77130 A-154772 asascauaGfaAfAfGfuuucuucaaaL96 1909 A-154773 VPusUfsugaAfgAfAfacuuUfcUfauguususa 2002 UAAACAUAGAAAGUUUCUUCAAC 2095 AD-77131 A-154774 asasccuaAfuUfAfGfuaacuuuccaL96 1910 A-154775 VPusGfsgaaAfgUfUfacuaAfuUfagguusgsc 2003 GCAACCUAAUUAGUAACUUUCCU 2096 AD-77132 A-154776 gsascuuaUfuUfCfCfuucacuuaaaL96 1911 A-154777 VPusUfsuaaGfuGfAfaggaAfaUfaagucsasu 2004 AUGACUUAUUUCCUUCACUUAAU 2097 AD-77133 A-154778 gsgscucuCfaAfAfCfuuagcaaaaaL96 1912 A-154779 VPusUfsuuuGfcUfAfaguuUfgAfgagccsasa 2005 UUGGCUCUCAAACUUAGCAAAAU 2098 AD-77134 A-154780 ascsaaagAfuAfAfGfugaaaagagaL96 1913 A-154781 VPusCfsucuUfuUfCfacuuAfuCfuuugusasu 2006 AUACAAAGAUAAGUGAAAAGAGA 2099 AD-77135 A-154782 gsusagugUfaCfUfGfuucaccaaaaL96 1914 A-154783 VPusUfsuugGfuGfAfacagUfaCfacuacsusu 2007 AAGUAGUGUACUGUUCACCAAAU 2100 AD-77136 A-154784 uscsaccaAfcUfCfAfuagcaaaguaL96 1915 A-154785 VPusAfscuuUfgCfUfaugaGfuUfggugasgsu 2008 ACUCACCAACUCAUAGCAAAGUC 2101 AD-77137 A-154786 asasaauaAfcUfCfAfuuauaccaaaL96 1916 A-154787 VPusUfsuggUfaUfAfaugaGfuUfauuuuscsa 2009 UGAAAAUAACUCAUUAUACCAAU 2102 AD-77138 A-154788 asasgaaaGfaAfUfCfuuuccauucaL96 1917 A-154789 VPusGfsaauGfgAfAfagauUfcUfuucuuscsu 2010 AGAAGAAAGAAUCUUUCCAUUCA 2103 AD-77139 A-154790 asusuucaAfgAfUfAfuuuguaaaaaL96 1918 A-154791 VPusUfsuuuAfcAfAfauauCfuUfgaaaususg 2011 CAAUUUCAAGAUAUUUGUAAAAG 2104 AD-77140 A-154792 asgscugaAfaCfUfCfuagaauuaaaL96 1919 A-154793 VPusUfsuaaUfuCfUfagagUfuUfcagcususg 2012 CAAGCUGAAACUCUAGAAUUAAA 2105 AD-77141 A-154794 asasagauAfaAfUfCfugauuuaugaL96 1920 A-154795 VPusCfsauaAfaUfCfagauUfuAfucuuususg 2013 CAAAAGAUAAAUCUGAUUUAUGC 2106 AD-77142 A-154796 usasuuaaAfuUfAfAfuucugauugaL96 1921 A-154797 VPusCfsaauCfaGfAfauuaAfuUfuaauasasa 2014 UUUAUUAAAUUAAUUCUGAUUGU 2107 AD-77143 A-154798 asasgaauGfaGfCfUfuucaacucaaL96 1922 A-154799 VPusUfsgagUfuGfAfaagcUfcAfuucuusasc 2015 GUAAGAAUGAGCUUUCAACUCAU 2108 AD-77144 A-154800 gsusuugaUfaAfCfAfuuuaaaagaaL96 1923 A-154801 VPusUfscuuUfuAfAfauguUfaUfcaaacsusu 2016 AAGUUUGAUAACAUUUAAAAGAU 2109 AD-77145 A-154802 usgsuuuuAfaAfCfAfuagaaaguuaL96 1924 A-154803 VPusAfsacuUfuCfUfauguUfuAfaaacasusa 2017 UAUGUUUUAAACAUAGAAAGUUU 2110 AD-77146 A-154804 usasaauaAfcCfCfAfuucauagcaaL96 1925 A-154805 VPusUfsgcuAfuGfAfauggGfuUfauuuasusa 2018 UAUAAAUAACCCAUUCAUAGCAU 2111 AD-77147 A-154806 csascauaGfaCfUfCfuuuaaaacuaL96 1926 A-154807 VPusAfsguuUfuAfAfagagUfcUfaugugsgsg 2019 CCCACAUAGACUCUUUAAAACUA 2112 AD-77148 A-154808 asasgucaCfaAfAfGfuuaucuucuaL96 1927 A-154809 VPusAfsgaaGfaUfAfacuuUfgUfgacuususu 2020 AAAAGUCACAAAGUUAUCUUCUU 2113 AD-77149 A-154810 usasaagaAfaGfAfAfuuuaugagaaL96 1928 A-154811 VPusUfscucAfuAfAfauucUfuUfcuuuasusc 2021 GAUAAAGAAAGAAUUUAUGAGAA 2114 AD-77150 A-154812 gsasaaugUfaUfGfUfuugacuuguaL96 1929 A-154813 VPusAfscaaGfuCfAfaacaUfaCfauuucsusa 2022 UAGAAAUGUAUGUUUGACUUGUU 2115 AD-77151 A-154814 ususuauaAfgAfCfCfuuccuguuaaL96 1930 A-154815 VPusUfsaacAfgGfAfagguCfuUfauaaasasu 2023 AUUUUAUAAGACCUUCCUGUUAG 2116 AD-77152 A-154816 csusgugaAfuGfUfGfuuuuauccaaL96 1931 A-154817 VPusUfsggaUfaAfAfacacAfuUfcacagsasg 2024 CUCUGUGAAUGUGUUUUAUCCAU 2117 AD-77153 A-154818 ususaauuCfuGfAfUfuguauuugaaL96 1932 A-154819 VPusUfscaaAfuAfCfaaucAfgAfauuaasusu 2025 AAUUAAUUCUGAUUGUAUUUGAA 2118 AD-77154 A-154820 usgsucauCfuAfCfCfuaccucaaaaL96 1933 A-154821 VPusUfsuugAfgGfUfagguAfgAfugacasusa 2026 UAUGUCAUCUACCUACCUCAAAG 2119 AD-77155 A-154822 csgsugaaAfgCfAfAfaacaauaggaL96 1934 A-154823 VPusCfscuaUfuGfUfuuugCfuUfucacgsusa 2027 UACGUGAAAGCAAAACAAUAGGG 2120 AD-77156 A-154824 asascaacAfaUfUfCfuauagauagaL96 1935 A-154825 VPusCfsuauCfuAfUfagaaUfuGfuuguususu 2028 AAAACAACAAUUCUAUAGAUAGA 2121 AD-77157 A-154826 usasaaguAfuUfAfUfuugaacuuuaL96 1936 A-154827 VPusAfsaagUfuCfAfaauaAfuAfcuuuasasu 2029 AUUAAAGUAUUAUUUGAACUUUU 2122 AD-77158 A-154828 asgsaaagAfaUfUfUfaugagaaauaL96 1937 A-154829 VPusAfsuuuCfuCfAfuaaaUfuCfuuucususu 2030 AAAGAAAGAAUUUAUGAGAAAUU 2123 AD-77159 A-154830 asasaugcAfcUfGfAfuguaaaguaaL96 1938 A-154831 VPusUfsacuUfuAfCfaucaGfuGfcauuususg 2031 CAAAAUGCACUGAUGUAAAGUAG 2124 AD-77160 A-154832 asusuaauUfcUfGfAfuuguauuugaL96 1939 A-154833 VPusCfsaaaUfaCfAfaucaGfaAfuuaaususu 2032 AAAUUAAUUCUGAUUGUAUUUGA 2125 AD-77161 A-154834 csasauaaUfgUfUfCfuauagaaaaaL96 1940 A-154835 VPusUfsuuuCfuAfUfagaaCfaUfuauugsasu 2033 AUCAAUAAUGUUCUAUAGAAAAG 2126 AD-77162 A-154836 gsasuuuuCfaAfUfUfugauuuugaaL96 1941 A-154837 VPusUfscaaAfaUfCfaaauUfgAfaaaucsasa 2034 UUGAUUUUCAAUUUGAUUUUGAA 2127 AD-77163 A-154840 ususgauuUfuGfAfAfuucugcauuaL96 1942 A-154841 VPusAfsaugCfaGfAfauucAfaAfaucaasasu 2035 AUUUGAUUUUGAAUUCUGCAUUU 2128 AD-77164 A-154842 ususucaaUfuUfGfAfuuuugaauuaL96 1943 A-154843 VPusAfsauuCfaAfAfaucaAfaUfugaaasasu 2036 AUUUUCAAUUUGAUUUUGAAUUC 2129 AD-77165 A-154844 gsusgucuGfuGfUfAfucaugaaaaaL96 1944 A-154845 VPusUfsuuuCfaUfGfauacAfcAfgacacsasg 2037 CUGUGUCUGUGUAUCAUGAAAAU 2130 AD-77166 A-154846 gsgsuuuuAfuGfAfAfuacaaagauaL96 1945 A-154847 VPusAfsucuUfuGfUfauucAfuAfaaaccsasa 2038 UUGGUUUUAUGAAUACAAAGAUA 2131 AD-77167 A-154848 gscsagauCfaAfGfAfuuuucucauaL96 1946 A-154849 VPusAfsugaGfaAfAfaucuUfgAfucugcsasg 2039 CUGCAGAUCAAGAUUUUCUCAUU 2132 AD-77168 A-154850 asascuaaUfuGfAfAfucauuaucuaL96 1947 A-154851 VPusAfsgauAfaUfGfauucAfaUfuaguusasc 2040 GUAACUAAUUGAAUCAUUAUCUU 2133 AD-77169 A-154852 usgsguuuAfuGfAfAfuuguuuccuaL96 1948 A-154853 VPusAfsggaAfaCfAfauucAfuAfaaccascsu 2041 AGUGGUUUAUGAAUUGUUUCCUU 2134 AD-77170 A-154854 usasguauAfaUfGfGfugcuauuuuaL96 1949 A-154855 VPusAfsaaaUfaGfCfaccaUfuAfuacuasasa 2042 UUUAGUAUAAUGGUGCUAUUUUG 2135 AD-77171 A-154856 csusguuuAfaUfAfAfgcauauuuuaL96 1950 A-154857 VPusAfsaaaUfaUfGfcuuaUfuAfaacagsusa 2043 UACUGUUUAAUAAGCAUAUUUUG 2136 AD-77172 A-154858 asasauauCfaAfAfAfccuuucaaaaL96 1951 A-154859 VPusUfsuugAfaAfGfguuuUfgAfuauuususg 2044 CAAAAUAUCAAAACCUUUCAAAU 2137 AD-77173 A-154860 ascsagauGfuAfAfAfagaaacuauaL96 1952 A-154861 VPusAfsuagUfuUfCfuuuuAfcAfucuguscsc 2045 GGACAGAUGUAAAAGAAACUAUA 2138 AD-77174 A-154862 asascuuuGfaGfGfCfcaaucauuuaL96 1953 A-154863 VPusAfsaauGfaUfUfggccUfcAfaaguusgsc 2046 GCAACUUUGAGGCCAAUCAUUUU 2139

Example 11—Further IGF-1 Transcripts, siRNA Design, and siRNA Screening Bioinformatics

A set of siRNAs targeting human IGF1 (human insulin like growth factor 1, NCBI refseqID: NM_000618; NCBI GeneID: 3479) were designed using custom R and Python scripts. The human IGF1 REFSEQ mRNA has a length of 7366 bases.

The rationale and method for the set of siRNA designs is as follows: the predicted efficacy for every potential 19mer siRNA from position 10 through the end was determined with a linear model derived the direct measure of mRNA knockdown from more than 20,000 distinct siRNA designs targeting a large number of vertebrate genes. The custom Python script built the set of siRNAs by systematically selecting a siRNA every 11 bases along the target mRNA starting at position 10. At each of the positions, the neighboring siRNA (one position to the 5′ end of the mRNA, one position to the 3′ end of the mRNA) was swapped into the design set if the predicted efficacy was better than the efficacy at the exact every-11th siRNA. Low complexity siRNAs, i.e., those with Shannon Entropy measures below 1.35 were excluded from the set.

In Vitro Dual-Glo® Screening Cell Culture and Transfections

Cos7 cells (ATCC, Manassas, Va.) were grown to near confluence at 37° C. in an atmosphere of 5% CO₂ in DMEM (ATCC) supplemented with 10% FBS, before being released from the plate by trypsinization. Three human IGF-1 Dual-Glo® Luciferase constructs were generated using the psiCHECK2 vector. Construct one contained sequence based on NM_001111285, while constructs two and three contained sequence based on NM_000618 as provided in the prior Example. Dual-luciferase plasmids were co-transfected with siRNA into 5000 cells using Lipofectamine RNAiMax (Invitrogen, Carlsbad Calif. cat #13778-150). For each well of a 384 well plate, 0.1 μl of Lipofectamine was added to 15 ng of plasmid vector and siRNA in 15 μl of Opti-MEM and allowed to complex at room temperature for 15 minutes. The mixture was then added to the cells resuspended in 35 ul of fresh complete media. Cells were incubated for 48 hours before luciferase was measured. Single dose experiments were performed at 10 nM final duplex concentration.

Dual-Glo® Luciferase Assay

Forty-eight hours after the siRNAs were transfected, Firefly (transfection control) and Renilla (fused to IGF1 target sequence in 3′ UTR) luciferase were measured. First, media was removed from cells. Then Firefly luciferase activity was measured by adding 20 ul of Dual-Glo® Luciferase Reagent mixed with 20 ul of complete media to each well. The mixture was incubated at room temperature for 30 minutes before luminescense (500 nm) was measured on a Spectramax (Molecular Devices) to detect the Firefly luciferase signal. Renilla luciferase activity was measured by adding 20 ul of room temperature of Dual-Glo® Stop & Glo® Reagent to each well and the plates were incubated for 20 minutes before luminescence was again measured to determine the Renilla luciferase signal. The Dual-Glo® Stop & Glo® Reagent quenched the firefly luciferase signal and sustained luminescence for the Renilla luciferase reaction. siRNA activity was determined by normalizing the Renilla (IGF1) signal to the Firefly (control) signal within each well. The magnitude of siRNA activity was then assessed relative to cells that were transfected with the same vector but were not treated with siRNA or were treated with a non-targeting siRNA. All transfections were done in quadruplicates.

TABLE 18 Unmodified Sense and Antisense Strand Sequences of IGF-1 dsRNAs Sense Position SEQ Antisense  Position in Duplex Oligo in NM_ ID Oligo NM_ SEQ ID Name Name Sense Sequence 000618.3 NO Name Antisense Sequence 000618.3 NO AD-74963 A-150432 UAGAUAAAUGUGAGGAUUU    6-24 2140 A-150433 AAAUCCUCACAUUUAUCUA    6-24 2420 AD-74964 A-150434 UUCUCUAAAUCCCUCUUCU   24-42 2141 A-150435 AGAAGAGGGAUUUAGAGAA   24-42 2421 AD-74965 A-150436 CUGUUUGCUAAAUCUCACU   41-59 2142 A-150437 AGUGAGAUUUAGCAAACAG   41-59 2422 AD-74966 A-150438 CUCACUGUCACUGCUAAAU   54-72 2143 A-150439 AUUUAGCAGUGACAGUGAG   54-72 2423 AD-74967 A-150440 UUCAGAGCAGAUAGAGCCU   72-90 2144 A-150441 AGGCUCUAUCUGCUCUGAA   72-90 2424 AD-74968 A-150442 CAUUGCUCUCAACAUCUCA  127-145 2145 A-150443 UGAGAUGUUGAGAGCAAUG  127-145 2425 AD-74969 A-150444 ACCAAUUCAUUUUCAGACU  185-203 2146 A-150445 AGUCUGAAAAUGAAUUGGU  185-203 2426 AD-74970 A-150446 UUUGUACUUCAGAAGCAAU  203-221 2147 A-150447 AUUGCUUCUGAAGUACAAA  203-221 2427 AD-74971 A-150448 AUGGGAAAAAUCAGCAGUA  220-238 2148 A-150449 UACUGCUGAUUUUUCCCAU  220-238 2428 AD-74972 A-150450 CAAUUAUUUAAGUGCUGCU  247-265 2149 A-150451 AGCAGCACUUAAAUAAUUG  247-265 2429 AD-74973 A-150452 UUGAAGGUGAAGAUGCACA  277-295 2150 A-150453 UGUGCAUCUUCACCUUCAA  277-295 2430 AD-74974 A-150454 UUUUAUUUCAACAAGCCCA  430-448 2151 A-150455 UGGGCUUGUUGAAAUAAAA  430-448 2431 AD-74975 A-150456 CACAGGGUAUGGCUCCAGA  447-465 2152 A-150457 UCUGGAGCCAUACCCUGUG  447-465 2432 AD-74976 A-150458 CAGCAGUCGGAGGGCGCCU  462-480 2153 A-150459 AGGCGCCCUCCGACUGCUG  462-480 2433 AD-74977 A-150460 UUGCGCACCCCUCAAGCCU  543-561 2154 A-150461 AGGCUUGAGGGGUGCGCAA  543-561 2434 AD-74978 A-150462 UGCAGGAAACAAGAACUAA  654-672 2155 A-150463 UUAGUUCUUGUUUCCUGCA  654-672 2435 AD-74979 A-150464 CAGGAUGUAGGAAGACCCU  672-690 2156 A-150465 AGGGUCUUCCUACAUCCUG  672-690 2436 AD-74980 A-150466 UUAAACUUUGGAACACCUA  750-768 2157 A-150467 UAGGUGUUCCAAAGUUUAA  750-768 2437 AD-74981 A-150468 AAAUAAGUUUGAUAACAUU  774-792 2158 A-150469 AAUGUUAUCAAACUUAUUU  774-792 2438 AD-74982 A-150470 UUAAAAGAUGGGCGUUUCA  792-810 2159 A-150471 UGAAACGCCCAUCUUUUAA  792-810 2439 AD-74983 A-150472 AAAUACACAAGUAAACAUU  818-836 2160 A-150473 AAUGUUUACUUGUGUAUUU  818-836 2440 AD-74984 A-150474 UUCCAACAUUGUCUUUAGA  835-853 2161 A-150475 UCUAAAGACAAUGUUGGAA  835-853 2441 AD-74985 A-150476 GGAGUGAUUUGCACCUUGA  852-870 2162 A-150477 UCAAGGUGCAAAUCACUCC  852-870 2442 AD-74986 A-150478 AUUGCUGUUGAUCUUUUAU  894-912 2163 A-150479 AUAAAAGAUCAACAGCAAU  894-912 2443 AD-74987 A-150480 UCAAUAAUGUUCUAUAGAA  912-930 2164 A-150481 UUCUAUAGAACAUUAUUGA  912-930 2444 AD-74988 A-150482 AAAGAAAAAAAAAAUAUAU  930-948 2165 A-150483 AUAUAUUUUUUUUUUCUUU  930-948 2445 AD-74989 A-150484 AUAUAUAUAUAUAUCUUAA  947-965 2166 A-150485 UUAAGAUAUAUAUAUAUAU  947-965 2446 AD-74990 A-150486 UUUCCUUAUUUGCACUUCU 1091-1109 2167 A-150487 AGAAGUGCAAAUAAGGAAA 1091-1109 2447 AD-74991 A-150488 CUUUCUACACAACUCGGGA 1108-1126 2168 A-150489 UCCCGAGUUGUGUAGAAAG 1108-1126 2448 AD-74992 A-150490 GCUGUUUGUUUUACAGUGU 1125-1143 2169 A-150491 ACACUGUAAAACAAACAGC 1125-1143 2449 AD-74993 A-150492 UUACAGUGUCUGAUAAUCU 1135-1153 2170 A-150493 AGAUUAUCAGACACUGUAA 1135-1153 2450 AD-74994 A-150494 CUGAUAAUCUUGUUAGUCU 1144-1162 2171 A-150495 AGACUAACAAGAUUAUCAG 1144-1162 2451 AD-74995 A-150496 UAUACCCACCACCUCCCUU 1162-1180 2172 A-150497 AAGGGAGGUGGUGGGUAUA 1162-1180 2452 AD-74996 A-150498 UUGCCGAAUUUGGCCUCCU 1195-1213 2173 A-150499 AGGAGGCCAAAUUCGGCAA 1195-1213 2453 AD-74997 A-150500 GCCGAAUUUGGCCUCCUCA 1197-1215 2174 A-150501 UGAGGAGGCCAAAUUCGGC 1197-1215 2454 AD-74998 A-150502 AAAAGCAGCAGCAAGUCGU 1215-1233 2175 A-150503 ACGACUUGCUGCUGCUUUU 1215-1233 2455 AD-74999 A-150504 GUCAAGAAGCACACCAAUU 1232-1250 2176 A-150505 AAUUGGUGUGCUUCUUGAC 1232-1250 2456 AD-75000 A-150506 AGUUGGAUGCAUUUUAUUU 1293-1311 2177 A-150507 AAAUAAAAUGCAUCCAACU 1293-1311 2457 AD-75001 A-150508 UUAGACACAAAGCUUUAUU 1311-1329 2178 A-150509 AAUAAAGCUUUGUGUCUAA 1311-1329 2458 AD-75002 A-150510 CACAUCAUGCUUACAAAAA 1334-1352 2179 A-150511 UUUUUGUAAGCAUGAUGUG 1334-1352 2459 AD-75003 A-150512 AAGAAUAAUGCAAAUAGUU 1352-1370 2180 A-150513 AACUAUUUGCAUUAUUCUU 1352-1370 2460 AD-75004 A-150514 UGCAACUUUGAGGCCAAUA 1370-1388 2181 A-150515 UAUUGGCCUCAAAGUUGCA 1370-1388 2461 AD-75005 A-150516 CAUUUUUAGGCAUAUGUUU 1388-1406 2182 A-150517 AAACAUAUGCCUAAAAAUG 1388-1406 2462 AD-75006 A-150518 UUAAACAUAGAAAGUUUCU 1406-1424 2183 A-150519 AGAAACUUUCUAUGUUUAA 1406-1424 2463 AD-75007 A-150520 CUUCAACUCAAAAGAGUUA 1423-1441 2184 A-150521 UAACUCUUUUGAGUUGAAG 1423-1441 2464 AD-75008 A-150522 UCCUUCAAAUGAUGAGUUA 1440-1458 2185 A-150523 UAACUCAUCAUUUGAAGGA 1440-1458 2465 AD-75009 A-150524 UUAGUAACUUUCCUCUUUU 1472-1490 2186 A-150525 AAAAGAGGAAAGUUACUAA 1472-1490 2466 AD-75010 A-150526 UUUUUCCAUAUAGAGCACU 1494-1512 2187 A-150527 AGUGCUCUAUAUGGAAAAA 1494-1512 2467 AD-75011 A-150528 CUAUGUAAAUUUAGCAUAU 1511-1529 2188 A-150529 AUAUGCUAAAUUUACAUAG 1511-1529 2468 AD-75012 A-150530 AUCAAUUAUACAGGAUAUA 1528-1546 2189 A-150531 UAUAUCCUGUAUAAUUGAU 1528-1546 2469 AD-75013 A-150532 UUUAGUAUAAUGGUGCUAU 1572-1590 2190 A-150533 AUAGCACCAUUAUACUAAA 1572-1590 2470 AD-75014 A-150534 UUGUUAUAUGAAAGAGUCU 1599-1617 2191 A-150535 AGACUCUUUCAUAUAACAA 1599-1617 2471 AD-75015 A-150536 ACGGUAAUACGUGAAAGCA 1625-1643 2192 A-150537 UGCUUUCACGUAUUACCGU 1625-1643 2472 AD-75016 A-150538 AAAACAAUAGGGGAAGCCU 1643-1661 2193 A-150539 AGGCUUCCCCUAUUGUUUU 1643-1661 2473 AD-75017 A-150540 UACUGAAAACACCAUCCAU 1690-1708 2194 A-150541 AUGGAUGGUGUUUUCAGUA 1690-1708 2474 AD-75018 A-150542 UUGGGAAAGAAGGCAAAGU 1709-1727 2195 A-150543 ACUUUGCCUUCUUUCCCAA 1709-1727 2475 AD-75019 A-150544 UCAGACACAAAAGUCCACU 1757-1775 2196 A-150545 AGUGGACUUUUGUGUCUGA 1757-1775 2476 AD-75020 A-150546 CGAGUCCAGAGAGGAAACU 1793-1811 2197 A-150547 AGUUUCCUCUCUGGACUCG 1793-1811 2477 AD-75021 A-150548 AAACUGUGGAAUGGAAAAA 1807-1825 2198 A-150549 UUUUUCCAUUCCACAGUUU 1807-1825 2478 AD-75022 A-150550 AGCAGAAGGCUAGGAAUUU 1825-1843 2199 A-150551 AAAUUCCUAGCCUUCUGCU 1825-1843 2479 AD-75023 A-150552 UUAGCAGUCCUGGUUUCUU 1843-1861 2200 A-150553 AAGAAACCAGGACUGCUAA 1843-1861 2480 AD-75024 A-150554 CAAAAUGGGGGCAAUAUGU 1966-1984 2201 A-150555 ACAUAUUGCCCCCAUUUUG 1966-1984 2481 AD-75025 A-150556 UUUAAAAAGAUAAAGAUUA 2016-2034 2202 A-150557 UAAUCUUUAUCUUUUUAAA 2016-2034 2482 AD-75026 A-150558 UCAGAUUUUUUUUACCCUA 2033-2051 2203 A-150559 UAGGGUAAAAAAAAUCUGA 2033-2051 2483 AD-75027 A-150560 UUUUUUACCCUGGGUUGCU 2040-2058 2204 A-150561 AGCAACCCAGGGUAAAAAA 2040-2058 2484 AD-75028 A-150562 CUGUAAGGGUGCAACAUCA 2057-2075 2205 A-150563 UGAUGUUGCACCCUUACAG 2057-2075 2485 AD-75029 A-150564 CUGAGAUGCAAGGAAUUCU 2090-2108 2206 A-150565 AGAAUUCCUUGCAUCUCAG 2090-2108 2486 AD-75030 A-150566 UUGGUGAAUUGAAUGCUCA 2140-2158 2207 A-150567 UGAGCAUUCAAUUCACCAA 2140-2158 2487 AD-75031 A-150568 UUCUUGUCAGUGAAGCUAU 2170-2188 2208 A-150569 AUAGCUUCACUGACAAGAA 2170-2188 2488 AD-75032 A-150570 AAUAACUGGCCAACUAGUU 2192-2210 2209 A-150571 AACUAGUUGGCCAGUUAUU 2192-2210 2489 AD-75033 A-150572 UGUUAAAAGCUAACAGCUA 2210-2228 2210 A-150573 UAGCUGUUAGCUUUUAACA 2210-2228 2490 AD-75034 A-150574 CAAUCUCUUAAAACACUUU 2228-2246 2211 A-150575 AAAGUGUUUUAAGAGAUUG 2228-2246 2491 AD-75035 A-150576 AAAAUAUGUGGGAAGCAUU 2249-2267 2212 A-150577 AAUGCUUCCCACAUAUUUU 2249-2267 2492 AD-75036 A-150578 UUUGAUUUUCAAUUUGAUU 2266-2284 2213 A-150579 AAUCAAAUUGAAAAUCAAA 2266-2284 2493 AD-75037 A-150580 UUGAAUUCUGCAUUUGGUU 2285-2303 2214 A-150581 AACCAAAUGCAGAAUUCAA 2285-2303 2494 AD-75038 A-150582 UUUAUGAAUACAAAGAUAA 2303-2321 2215 A-150583 UUAUCUUUGUAUUCAUAAA 2303-2321 2495 AD-75039 A-150584 GUGAAAAGAGAGAAAGGAA 2322-2340 2216 A-150585 UUCCUUUCUCUCUUUUCAC 2322-2340 2496 AD-75040 A-150586 AAAGAAAAAGGAGAAAAAC 2340-2358 2217 A-150587 GUUUUUCUCCUUUUUCUUU 2340-2358 2497 AD-75041 A-150588 ACAAAGAGAUUUCUACCAA 2357-2375 2218 A-150589 UUGGUAGAAAUCUCUUUGU 2357-2375 2498 AD-75042 A-150590 UUGUUAGCACUCACUGACU 2398-2416 2219 A-150591 AGUCAGUGAGUGCUAACAA 2398-2416 2499 AD-75043 A-150592 UACAUAUCUAGUAAAACCU 2432-2450 2220 A-150593 AGGUUUUACUAGAUAUGUA 2432-2450 2500 AD-75044 A-150594 CUCGUUUAAUACUAUAAAU 2449-2467 2221 A-150595 AUUUAUAGUAUUAAACGAG 2449-2467 2501 AD-75045 A-150596 UUUAAUACUAUAAAUAAUA 2453-2471 2222 A-150597 UAUUAUUUAUAGUAUUAAA 2453-2471 2502 AD-75046 A-150598 UAUUCUAUUCAUUUUGAAA 2470-2488 2223 A-150599 UUUCAAAAUGAAUAGAAUA 2470-2488 2503 AD-75047 A-150600 UUUGAAAAACACAAUGAUU 2482-2500 2224 A-150601 AAUCAUUGUGUUUUUCAAA 2482-2500 2504 AD-75048 A-150602 AAGGAAAGUGAUCCAAAAU 2521-2539 2225 A-150603 AUUUUGGAUCACUUUCCUU 2521-2539 2505 AD-75049 A-150604 UUUGAAAUAUUAAAAUAAU 2539-2557 2226 A-150605 AUUAUUUUAAUAUUUCAAA 2539-2557 2506 AD-75050 A-150606 UUAAAAUAAUAUCUAAUAA 2548-2566 2227 A-150607 UUAUUAGAUAUUAUUUUAA 2548-2566 2507 AD-75051 A-150608 AAAAGUCACAAAGUUAUCU 2566-2584 2228 A-150609 AGAUAACUUUGUGACUUUU 2566-2584 2508 AD-75052 A-150610 UUCUUUAACAAACUUUACU 2584-2602 2229 A-150611 AGUAAAGUUUGUUAAAGAA 2584-2602 2509 AD-75053 A-150612 CUCUUAUUCUUAGCUGUAU 2601-2619 2230 A-150613 AUACAGCUAAGAAUAAGAG 2601-2619 2510 AD-75054 A-150614 AUAUACAUUUUUUUAAAAG 2618-2636 2231 A-150615 CUUUUAAAAAAAUGUAUAU 2618-2636 2511 AD-75055 A-150616 CAUUUUUUUAAAAGUUUGU 2623-2641 2232 A-150617 ACAAACUUUUAAAAAAAUG 2623-2641 2512 AD-75056 A-150618 GUUAAAAUAUGCUUGACUA 2640-2658 2233 A-150619 UAGUCAAGCAUAUUUUAAC 2640-2658 2513 AD-75057 A-150620 AUGCUUGACUAGAGUUUCA 2648-2666 2234 A-150621 UGAAACUCUAGUCAAGCAU 2648-2666 2514 AD-75058 A-150622 CAGUUGAAAGGCAAAAACU 2666-2684 2235 A-150623 AGUUUUUGCCUUUCAACUG 2666-2684 2515 AD-75059 A-150624 UUCCAUCACAACAAGAAAU 2684-2702 2236 A-150625 AUUUCUUGUUGUGAUGGAA 2684-2702 2516 AD-75060 A-150626 UUGGUAUCAAGAAAGUCCA 2771-2789 2237 A-150627 UGGACUUUCUUGAUACCAA 2771-2789 2517 AD-75061 A-150628 GUUAGUGUACUAGUCCAUA 2793-2811 2238 A-150629 UAUGGACUAGUACACUAAC 2793-2811 2518 AD-75062 A-150630 CAUAGCCUAGAAAAUGAUA 2811-2829 2239 A-150631 UAUCAUUUUCUAGGCUAUG 2811-2829 2519 AD-75063 A-150632 UCCCUAUCUGCAGAUCAAA 2828-2846 2240 A-150633 UUUGAUCUGCAGAUAGGGA 2828-2846 2520 AD-75064 A-150634 UUAUCCAGCAUUCAGAUCU 2869-2887 2241 A-150635 AGAUCUGAAUGCUGGAUAA 2869-2887 2521 AD-75065 A-150636 UUUUUGGUUAAAAGUACCA 2906-2924 2242 A-150637 UGGUACUUUUAACCAAAAA 2906-2924 2522 AD-75066 A-150638 UACCCAGGCUUGAUUAUUU 2920-2938 2243 A-150639 AAAUAAUCAAGCCUGGGUA 2920-2938 2523 AD-75067 A-150640 UCAUGCAAAUUCUAUAUUU 2938-2956 2244 A-150641 AAAUAUAGAAUUUGCAUGA 2938-2956 2524 AD-75068 A-150642 UUACAUUCUUGGAAAGUCU 2956-2974 2245 A-150643 AGACUUUCCAAGAAUGUAA 2956-2974 2525 AD-75069 A-150644 UCUUGGAAAGUCUAUAUGA 2962-2980 2246 A-150645 UCAUAUAGACUUUCCAAGA 2962-2980 2526 AD-75070 A-150646 AAAAACAAAAAUAACAUCU 2980-2998 2247 A-150647 AGAUGUUAUUUUUGUUUUU 2980-2998 2527 AD-75071 A-150648 UUCUCCCACUGGGUCACCU 3006-3024 2248 A-150649 AGGUGACCCAGUGGGAGAA 3006-3024 2528 AD-75072 A-150650 CAAGGAUCAGAGGCCAGGA 3025-3043 2249 A-150651 UCCUGGCCUCUGAUCCUUG 3025-3043 2529 AD-75073 A-150652 AAAAAAAAAAAAAAGACUA 3043-3061 2250 A-150653 UAGUCUUUUUUUUUUUUUU 3043-3061 2530 AD-75074 A-150654 UCCCUGGAUCUCUGAAUAU 3060-3078 2251 A-150655 AUAUUCAGAGAUCCAGGGA 3060-3078 2531 AD-75075 A-150656 AUAUGCAAAAAGAAGGCCA 3077-3095 2252 A-150657 UGGCCUUCUUUUUGCAUAU 3077-3095 2532 AD-75076 A-150658 UAGUGGAGCCAGCAAUCCU 3100-3118 2253 A-150659 AGGAUUGCUGGCUCCACUA 3100-3118 2533 AD-75077 A-150660 UUAACUCUCAGUCCAACAU 3137-3155 2254 A-150661 AUGUUGGACUGAGAGUUAA 3137-3155 2534 AD-75078 A-150662 UUAUUUGAAUUGAGCACCU 3155-3173 2255 A-150663 AGGUGCUCAAUUCAAAUAA 3155-3173 2535 AD-75079 A-150664 CAGAUGUAAAAGAAACUAU 3208-3226 2256 A-150665 AUAGUUUCUUUUACAUCUG 3208-3226 2536 AD-75080 A-150666 AUACAUCAUUUUUGCCCUA 3225-3243 2257 A-150667 UAGGGCAAAAAUGAUGUAU 3225-3243 2537 AD-75081 A-150668 UUUUGCCCUCUGCCUGUUU 3234-3252 2258 A-150669 AAACAGGCAGAGGGCAAAA 3234-3252 2538 AD-75082 A-150670 UUCCAGACAUACAGGUUCU 3252-3270 2259 A-150671 AGAACCUGUAUGUCUGGAA 3252-3270 2539 AD-75083 A-150672 CUGUGGAAUAAGAUACUGA 3269-3287 2260 A-150673 UCAGUAUCUUAUUCCACAG 3269-3287 2540 AD-75084 A-150674 UAAGAUACUGGACUCCUCU 3277-3295 2261 A-150675 AGAGGAGUCCAGUAUCUUA 3277-3295 2541 AD-75085 A-150676 CUUCCCAAGAUGGCACUUA 3294-3312 2262 A-150677 UAAGUGCCAUCUUGGGAAG 3294-3312 2542 AD-75086 A-150678 GUGUACCUUUUAAAAUUAU 3334-3352 2263 A-150679 AUAAUUUUAAAAGGUACAC 3334-3352 2543 AD-75087 A-150680 UUCCCUCUCAACAAAACUU 3352-3370 2264 A-150681 AAGUUUUGUUGAGAGGGAA 3352-3370 2544 AD-75088 A-150682 UUUAUAGGCAGUCUUCUGA 3369-3387 2265 A-150683 UCAGAAGACUGCCUAUAAA 3369-3387 2545 AD-75089 A-150684 UUUUCUGUCAUAGUUAGAU 3400-3418 2266 A-150685 AUCUAACUAUGACAGAAAA 3400-3418 2546 AD-75090 A-150686 AUGUGAUAAUUCUAAGAGU 3417-3435 2267 A-150687 ACUCUUAGAAUUAUCACAU 3417-3435 2547 AD-75091 A-150688 UUCCUUCACUUAAUUCUAU 3449-3467 2268 A-150689 AUAGAAUUAAGUGAAGGAA 3449-3467 2548 AD-75092 A-150690 AUUAUCUUUCUUAACUUUU 3517-3535 2269 A-150691 AAAAGUUAAGAAAGAUAAU 3517-3535 2549 AD-75093 A-150692 UUCCAACACAUAAUCCUCU 3535-3553 2270 A-150693 AGAGGAUUAUGUGUUGGAA 3535-3553 2550 AD-75094 A-150694 AAAUAAAUUGAAAAUAACU 3567-3585 2271 A-150695 AGUUAUUUUCAAUUUAUUU 3567-3585 2551 AD-75095 A-150696 UCAUUAUACCAAUUCACUA 3585-3603 2272 A-150697 UAGUGAAUUGGUAUAAUGA 3585-3603 2552 AD-75096 A-150698 AUUUUAUUUUUUAAUGAAU 3603-3621 2273 A-150699 AUUCAUUAAAAAAUAAAAU 3603-3621 2553 AD-75097 A-150700 UUAAAACUAGAAAACAAAU 3621-3639 2274 A-150701 AUUUGUUUUCUAGUUUUAA 3621-3639 2554 AD-75098 A-150702 UUGAUUACUAUAUACUACA 3662-3680 2275 A-150703 UGUAGUAUAUAGUAAUCAA 3662-3680 2555 AD-75099 A-150704 AUGACUCAGAUUUCAUAGA 3686-3704 2276 A-150705 UCUAUGAAAUCUGAGUCAU 3686-3704 2556 AD-75100 A-150706 AAAGGAGCAACCAAAAUGU 3704-3722 2277 A-150707 ACAUUUUGGUUGCUCCUUU 3704-3722 2557 AD-75101 A-150708 GUCACAACCCAAAACUUUA 3721-3739 2278 A-150709 UAAAGUUUUGGGUUGUGAC 3721-3739 2558 AD-75102 A-150710 AAACUUUACAAGCUUUGCU 3732-3750 2279 A-150711 AGCAAAGCUUGUAAAGUUU 3732-3750 2559 AD-75103 A-150712 UUCAGAAUUAGAUUGCUUU 3750-3768 2280 A-150713 AAAGCAAUCUAAUUCUGAA 3750-3768 2560 AD-75104 A-150714 UUAUAAUUCUUGAAUGAGA 3767-3785 2281 A-150715 UCUCAUUCAAGAAUUAUAA 3767-3785 2561 AD-75105 A-150716 UAAUUCUUGAAUGAGGCAA 3770-3788 2282 A-150717 UUGCCUCAUUCAAGAAUUA 3770-3788 2562 AD-75106 A-150718 AUUUCAAGAUAUUUGUAAA 3788-3806 2283 A-150719 UUUACAAAUAUCUUGAAAU 3788-3806 2563 AD-75107 A-150720 AAGAACAGUAAACAUUGGU 3806-3824 2284 A-150721 ACCAAUGUUUACUGUUCUU 3806-3824 2564 AD-75108 A-150722 UUUCAACUCAUAGGCUUAU 3835-3853 2285 A-150723 AUAAGCCUAUGAGUUGAAA 3835-3853 2565 AD-75109 A-150724 UUGACCAUACUGGAUACUU 3865-3883 2286 A-150725 AAGUAUCCAGUAUGGUCAA 3865-3883 2566 AD-75110 A-150726 UUUAAGAUGAGGCAGUUCA 3939-3957 2287 A-150727 UGAACUGCCUCAUCUUAAA 3939-3957 2567 AD-75111 A-150728 CAUCAGAAUCCACUCUUCU 3982-4000 2288 A-150729 AGAAGAGUGGAUUCUGAUG 3982-4000 2568 AD-75112 A-150730 UAGGGAUAUGAAAAUCUCU 4000-4018 2289 A-150731 AGAGAUUUUCAUAUCCCUA 4000-4018 2569 AD-75113 A-150732 UUCACCCUAAGGAUCCAAU 4081-4099 2290 A-150733 AUUGGAUCCUUAGGGUGAA 4081-4099 2570 AD-75114 A-150734 AUGGAAUACUGAAAAGAAA 4098-4116 2291 A-150735 UUUCUUUUCAGUAUUCCAU 4098-4116 2571 AD-75115 A-150736 GAAUACUGAAAAGAAAUCA 4101-4119 2292 A-150737 UGAUUUCUUUUCAGUAUUC 4101-4119 2572 AD-75116 A-150738 ACUUCCUUGAAAAUUUUAU 4119-4137 2293 A-150739 AUAAAAUUUUCAAGGAAGU 4119-4137 2573 AD-75117 A-150740 UUAAAAAACAAACAAACAA 4137-4155 2294 A-150741 UUGUUUGUUUGUUUUUUAA 4137-4155 2574 AD-75118 A-150742 AAACAAAAAGCCUGUCCAA 4154-4172 2295 A-150743 UUGGACAGGCUUUUUGUUU 4154-4172 2575 AD-75119 A-150744 UUUGUGUAGAUGAAACCAU 4208-4226 2296 A-150745 AUGGUUUCAUCUACACAAA 4208-4226 2576 AD-75120 A-150746 UUGGGAGAAGGCUUAGAAU 4271-4289 2297 A-150747 AUUCUAAGCCUUCUCCCAA 4271-4289 2577 AD-75121 A-150748 UAAAAGAUGUAGCACAUUU 4289-4307 2298 A-150749 AAAUGUGCUACAUCUUUUA 4289-4307 2578 AD-75122 A-150750 UUAUUGUUUGGCCAGCUAU 4319-4337 2299 A-150751 AUAGCUGGCCAAACAAUAA 4319-4337 2579 AD-75123 A-150752 AUGCCAAUGUGGUGCUAUU 4336-4354 2300 A-150753 AAUAGCACCACAUUGGCAU 4336-4354 2580 AD-75124 A-150754 AAUGUGGUGCUAUUGUUUA 4341-4359 2301 A-150755 UAAACAAUAGCACCACAUU 4341-4359 2581 AD-75125 A-150756 CUUUAAGAAAGUACUUGAA 4359-4377 2302 A-150757 UUCAAGUACUUUCUUAAAG 4359-4377 2582 AD-75126 A-150758 CUAAAAAAAAAAGAAAAAA 4377-4395 2303 A-150759 UUUUUUCUUUUUUUUUUAG 4377-4395 2583 AD-75127 A-150760 AAGAAAAAAAAGAAAGCAU 4395-4413 2304 A-150761 AUGCUUUCUUUUUUUUCUU 4395-4413 2584 AD-75128 A-150762 AUAGACAUAUUUUUUUAAA 4412-4430 2305 A-150763 UUUAAAAAAAUAUGUCUAU 4412-4430 2585 AD-75129 A-150764 UUAAAGUAUAAAAACAACA 4426-4444 2306 A-150765 UGUUGUUUUUAUACUUUAA 4426-4444 2586 AD-75130 A-150766 CAAUUCUAUAGAUAGAUGA 4443-4461 2307 A-150767 UCAUCUAUCUAUAGAAUUG 4443-4461 2587 AD-75131 A-150768 GGCUUAAUAAAAUAGCAUU 4460-4478 2308 A-150769 AAUGCUAUUUUAUUAAGCC 4460-4478 2588 AD-75132 A-150770 UAAUAAAAUAGCAUUAGGU 4464-4482 2309 A-150771 ACCUAAUGCUAUUUUAUUA 4464-4482 2589 AD-75133 A-150772 UAUCUAGCCACCACCACCU 4484-4502 2310 A-150773 AGGUGGUGGUGGCUAGAUA 4484-4502 2590 AD-75134 A-150774 UUUAUCACUCACAAGUAGU 4511-4529 2311 A-150775 ACUACUUGUGAGUGAUAAA 4511-4529 2591 AD-75135 A-150776 GGCAGGAGUUGGAAAUUUU 4566-4584 2312 A-150777 AAAAUUUCCAACUCCUGCC 4566-4584 2592 AD-75136 A-150778 UUUAAAGUUAGAAGGCUCA 4584-4602 2313 A-150779 UGAGCCUUCUAACUUUAAA 4584-4602 2593 AD-75137 A-150780 CCAUUGUUUUGUUGGCUCU 4601-4619 2314 A-150781 AGAGCCAACAAAACAAUGG 4601-4619 2594 AD-75138 A-150782 UUAGCAAAAUUAGCAAUAU 4625-4643 2315 A-150783 AUAUUGCUAAUUUUGCUAA 4625-4643 2595 AD-75139 A-150784 AUAUUAUCCAAUCUUCUGA 4642-4660 2316 A-150785 UCAGAAGAUUGGAUAAUAU 4642-4660 2596 AD-75140 A-150786 UUAUCCAAUCUUCUGAACU 4645-4663 2317 A-150787 AGUUCAGAAGAUUGGAUAA 4645-4663 2597 AD-75141 A-150788 AAGAGCAUGGAGAAUAAAC 4669-4687 2318 A-150789 GUUUAUUCUCCAUGCUCUU 4669-4687 2598 AD-75142 A-150790 ACGCGGGAAAAAAGAUCUU 4686-4704 2319 A-150791 AAGAUCUUUUUUCCCGCGU 4686-4704 2599 AD-75143 A-150792 GAUCUUAUAGGCAAAUAGA 4699-4717 2320 A-150793 UCUAUUUGCCUAUAAGAUC 4699-4717 2600 AD-75144 A-150794 AAGAAUUUAAAAGAUAAGU 4717-4735 2321 A-150795 ACUUAUCUUUUAAAUUCUU 4717-4735 2601 AD-75145 A-150796 GUAAGUUCCUUAUUGAUUU 4734-4752 2322 A-150797 AAAUCAAUAAGGAACUUAC 4734-4752 2602 AD-75146 A-150798 UUUUGUGCACUCUGCUCUA 4751-4769 2323 A-150799 UAGAGCAGAGUGCACAAAA 4751-4769 2603 AD-75147 A-150800 AAACAGAUAUUCAGCAAGU 4770-4788 2324 A-150801 ACUUGCUGAAUAUCUGUUU 4770-4788 2604 AD-75148 A-150802 UCAGCAAGUGGAGAAAAUA 4780-4798 2325 A-150803 UAUUUUCUCCACUUGCUGA 4780-4798 2605 AD-75149 A-150804 AAGAACAAAGAGAAAAAAU 4798-4816 2326 A-150805 AUUUUUUCUCUUUGUUCUU 4798-4816 2606 AD-75150 A-150806 AUACAUAGAUUUACCUGCA 4815-4833 2327 A-150807 UGCAGGUAAAUCUAUGUAU 4815-4833 2607 AD-75151 A-150808 UUACCUGCAAAAAAUAGCU 4825-4843 2328 A-150809 AGCUAUUUUUUGCAGGUAA 4825-4843 2608 AD-75152 A-150810 UUUAUAGAAGACAUUCUCA 4884-4902 2329 A-150811 UGAGAAUGUCUUCUAUAAA 4884-4902 2609 AD-75153 A-150812 AGACAUCUCAAAGAGCAGU 4911-4929 2330 A-150813 ACUGCUCUUUGAGAUGUCU 4911-4929 2610 AD-75154 A-150814 UAUGAGAUGGGGGUUAUCU 4987-5005 2331 A-150815 AGAUAACCCCCAUCUCAUA 4987-5005 2611 AD-75155 A-150816 CUACUGAUAAAGAAAGAAU 5004-5022 2332 A-150817 AUUCUUUCUUUAUCAGUAG 5004-5022 2612 AD-75156 A-150818 AAAGAAUUUAUGAGAAAUU 5016-5034 2333 A-150819 AAUUUCUCAUAAAUUCUUU 5016-5034 2613 AD-75157 A-150820 UAACAAUCUGUGAAGAUUU 5050-5068 2334 A-150821 AAAUCUUCACAGAUUGUUA 5050-5068 2614 AD-75158 A-150822 UUUUACUUUAUACAGUCUU 5098-5116 2335 A-150823 AAGACUGUAUAAAGUAAAA 5098-5116 2615 AD-75159 A-150824 UUUAUGAAUUUCUUAAUGU 5115-5133 2336 A-150825 ACAUUAAGAAAUUCAUAAA 5115-5133 2616 AD-75160 A-150826 UUAAUGUUCAAAAUGACUU 5127-5145 2337 A-150827 AAGUCAUUUUGAACAUUAA 5127-5145 2617 AD-75161 A-150828 UUCUUCUUUUUUUAUAUCA 5153-5171 2338 A-150829 UGAUAUAAAAAAAGAAGAA 5153-5171 2618 AD-75162 A-150830 AGAAUGAGGAAUAAUAAGU 5171-5189 2339 A-150831 ACUUAUUAUUCCUCAUUCU 5171-5189 2619 AD-75163 A-150832 UUAAACCCACAUAGACUCU 5189-5207 2340 A-150833 AGAGUCUAUGUGGGUUUAA 5189-5207 2620 AD-75164 A-150834 CUUUAAAACUAUAGGCUAA 5206-5224 2341 A-150835 UUAGCCUAUAGUUUUAAAG 5206-5224 2621 AD-75165 A-150836 AGAUAGAAAUGUAUGUUUA 5223-5241 2342 A-150837 UAAACAUACAUUUCUAUCU 5223-5241 2622 AD-75166 A-150838 UUUGACUUGUUGAAGCUAU 5238-5256 2343 A-150839 AUAGCUUCAACAAGUCAAA 5238-5256 2623 AD-75167 A-150840 UUUUUAAUCUUAAAAGAUU 5284-5302 2344 A-150841 AAUCUUUUAAGAUUAAAAA 5284-5302 2624 AD-75168 A-150842 UUGUGCUAAUUUAUUAGAA 5301-5319 2345 A-150843 UUCUAAUAAAUUAGCACAA 5301-5319 2625 AD-75169 A-150844 UUAUUAGAGCAGAACCUGU 5311-5329 2346 A-150845 ACAGGUUCUGCUCUAAUAA 5311-5329 2626 AD-75170 A-150846 GUUUGGCUCUCCUCAGAAA 5328-5346 2347 A-150847 UUUCUGAGGAGAGCCAAAC 5328-5346 2627 AD-75171 A-150848 CAAUAUUUUCAAAAGAUAA 5383-5401 2348 A-150849 UUAUCUUUUGAAAAUAUUG 5383-5401 2628 AD-75172 A-150850 UCAAAAGAUAAAUCUGAUU 5391-5409 2349 A-150851 AAUCAGAUUUAUCUUUUGA 5391-5409 2629 AD-75173 A-150852 UUAUGCAAUGGCAUCAUUU 5409-5427 2350 A-150853 AAAUGAUGCCAUUGCAUAA 5409-5427 2630 AD-75174 A-150854 UGCAAUGGCAUCAUUUAUU 5412-5430 2351 A-150855 AAUAAAUGAUGCCAUUGCA 5412-5430 2631 AD-75175 A-150856 UUUAAAACAGAAGAAUUGU 5430-5448 2352 A-150857 ACAAUUCUUCUGUUUUAAA 5430-5448 2632 AD-75176 A-150858 AACAACAAAAGGAAAAUGU 5505-5523 2353 A-150859 ACAUUUUCCUUUUGUUGUU 5505-5523 2633 AD-75177 A-150860 UUAAUCCUGUAGUACAUAU 5570-5588 2354 A-150861 AUAUGUACUACAGGAUUAA 5570-5588 2634 AD-75178 A-150862 UUUAAUAUUUUAUAAGACA 5603-5621 2355 A-150863 UGUCUUAUAAAAUAUUAAA 5603-5621 2635 AD-75179 A-150864 CCUUCCUGUUAGGUAUUAA 5620-5638 2356 A-150865 UUAAUACCUAACAGGAAGG 5620-5638 2636 AD-75180 A-150866 UUAGGUAUUAGAAAGUGAU 5628-5646 2357 A-150867 AUCACUUUCUAAUACCUAA 5628-5646 2637 AD-75181 A-150868 AUACAUAGAUAUCUUUUUU 5645-5663 2358 A-150869 AAAAAAGAUAUCUAUGUAU 5645-5663 2638 AD-75182 A-150870 UUUUUGUGUAAUUUCUAUU 5659-5677 2359 A-150871 AAUAGAAAUUACACAAAAA 5659-5677 2639 AD-75183 A-150872 UUAAAAAAGAGAGAAGACU 5677-5695 2360 A-150873 AGUCUUCUCUCUUUUUUAA 5677-5695 2640 AD-75184 A-150874 CUGUCAGAAGCUUUAAGUA 5694-5712 2361 A-150875 UACUUAAAGCUUCUGACAG 5694-5712 2641 AD-75185 A-150876 UAUGGUACAGGAUAAAGAU 5715-5733 2362 A-150877 AUCUUUAUCCUGUACCAUA 5715-5733 2642 AD-75186 A-150878 UUAAAUAACCAAUUCCUAU 5740-5758 2363 A-150879 AUAGGAAUUGGUUAUUUAA 5740-5758 2643 AD-75187 A-150880 UUGUUUUUUAAAGAAACCU 5773-5791 2364 A-150881 AGGUUUCUUUAAAAAACAA 5773-5791 2644 AD-75188 A-150882 CUCUCACAGAUAAGACAGA 5790-5808 2365 A-150883 UCUGUCUUAUCUGUGAGAG 5790-5808 2645 AD-75189 A-150884 CAGAAUUUUAUAGAGGGCU 5884-5902 2366 A-150885 AGCCCUCUAUAAAAUUCUG 5884-5902 2646 AD-75190 A-150886 UCUAGAAUUAAAGGAACCU 5917-5935 2367 A-150887 AGGUUCCUUUAAUUCUAGA 5917-5935 2647 AD-75191 A-150888 CUCACUGAAAACAUAUAUU 5934-5952 2368 A-150889 AAUAUAUGUUUUCAGUGAG 5934-5952 2648 AD-75192 A-150890 AAACAUAUAUUUCACGUGU 5942-5960 2369 A-150891 ACACGUGAAAUAUAUGUUU 5942-5960 2649 AD-75193 A-150892 GUUCCCUCUUUUUUUUUUU 5959-5977 2370 A-150893 AAAAAAAAAAAGAGGGAAC 5959-5977 2650 AD-75194 A-150894 UUAAGCGAUUCUCCUGCCU 6066-6084 2371 A-150895 AGGCAGGAGAAUCGCUUAA 6066-6084 2651 AD-75195 A-150896 CGGCUAAUUUUUUGGAUUU 6129-6147 2372 A-150897 AAAUCCAAAAAAUUAGCCG 6129-6147 2652 AD-75196 A-150898 UUUAAUAGAGACGGGGUUU 6147-6165 2373 A-150899 AAACCCCGUCUCUAUUAAA 6147-6165 2653 AD-75197 A-150900 UUUACCAUGUUGGCCAGGU 6164-6182 2374 A-150901 ACCUGGCCAACAUGGUAAA 6164-6182 2654 AD-75198 A-150902 UUGCUGGGAUUACAGGCAU 6231-6249 2375 A-150903 AUGCCUGUAAUCCCAGCAA 6231-6249 2655 AD-75199 A-150904 UUAAACAUGAUCCUUCUCU 6303-6321 2376 A-150905 AGAGAAGGAUCAUGUUUAA 6303-6321 2656 AD-75200 A-150906 GGGGUCUUUCAAGGGGAAA 6346-6364 2377 A-150907 UUUCCCCUUGAAAGACCCC 6346-6364 2657 AD-75201 A-150908 AAAAAUCCAAGCUUUUUUA 6364-6382 2378 A-150909 UAAAAAAGCUUGGAUUUUU 6364-6382 2658 AD-75202 A-150910 AAAGUAAAAAAAAAAAAAG 6382-6400 2379 A-150911 CUUUUUUUUUUUUUACUUU 6382-6400 2659 AD-75203 A-150912 AGAGAGGACACAAAACCAA 6399-6417 2380 A-150913 UUGGUUUUGUGUCCUCUCU 6399-6417 2660 AD-75204 A-150914 UUAAGAUGGAGACAGAGUU 6444-6462 2381 A-150915 AACUCUGUCUCCAUCUUAA 6444-6462 2661 AD-75205 A-150916 UUUCUCCUAAUAACCGGAA 6461-6479 2382 A-150917 UUCCGGUUAUUAGGAGAAA 6461-6479 2662 AD-75206 A-150918 GCUGAAUUACCUUUCACUU 6479-6497 2383 A-150919 AAGUGAAAGGUAAUUCAGC 6479-6497 2663 AD-75207 A-150920 UUCAAAAACAUGACCUUCA 6497-6515 2384 A-150921 UGAAGGUCAUGUUUUUGAA 6497-6515 2664 AD-75208 A-150922 CAAUCCUUAGAAUCUGCCU 6517-6535 2385 A-150923 AGGCAGAUUCUAAGGAUUG 6517-6535 2665 AD-75209 A-150924 UUUUAUAUUACUGAGGCCU 6538-6556 2386 A-150925 AGGCCUCAGUAAUAUAAAA 6538-6556 2666 AD-75210 A-150926 AAAAGUAAACAUUACUCAU 6557-6575 2387 A-150927 AUGAGUAAUGUUUACUUUU 6557-6575 2667 AD-75211 A-150928 UUUAUUUUGCCCAAAAUGA 6576-6594 2388 A-150929 UCAUUUUGGGCAAAAUAAA 6576-6594 2668 AD-75212 A-150930 CACUGAUGUAAAGUAGGAA 6594-6612 2389 A-150931 UUCCUACUUUACAUCAGUG 6594-6612 2669 AD-75213 A-150932 AAAAUAAAAACAGAGCUCU 6612-6630 2390 A-150933 AGAGCUCUGUUUUUAUUUU 6612-6630 2670 AD-75214 A-150934 CUAAAAUCCCUUUCAAGCA 6629-6647 2391 A-150935 UGCUUGAAAGGGAUUUUAG 6629-6647 2671 AD-75215 A-150936 UUGACCCCACUCACCAACU 6653-6671 2392 A-150937 AGUUGGUGAGUGGGGUCAA 6653-6671 2672 AD-75216 A-150938 UUAUCUUGUACCCGCUGCU 6722-6740 2393 A-150939 AGCAGCGGGUACAAGAUAA 6722-6740 2673 AD-75217 A-150940 CUGAAACCUCAAGCUGUCU 6762-6780 2394 A-150941 AGACAGCUUGAGGUUUCAG 6762-6780 2674 AD-75218 A-150942 GUAUCAUGAAAAUGUCUAU 6806-6824 2395 A-150943 AUAGACAUUUUCAUGAUAC 6806-6824 2675 AD-75219 A-150944 UUCAAAAUAUCAAAACCUU 6824-6842 2396 A-150945 AAGGUUUUGAUAUUUUGAA 6824-6842 2676 AD-75220 A-150946 UUUCAAAUAUCACGCAGCU 6841-6859 2397 A-150947 AGCUGCGUGAUAUUUGAAA 6841-6859 2677 AD-75221 A-150948 CUUAUAUUCAGUUUACAUA 6858-6876 2398 A-150949 UAUGUAAACUGAAUAUAAG 6858-6876 2678 AD-75222 A-150950 UUACAUAAAGGCCCCAAAU 6870-6888 2399 A-150951 AUUUGGGGCCUUUAUGUAA 6870-6888 2679 AD-75223 A-150952 AUACCAUGUCAGAUCUUUU 6887-6905 2400 A-150953 AAAAGAUCUGACAUGGUAU 6887-6905 2680 AD-75224 A-150954 AAAAGAGUUAAUGAACUAU 6910-6928 2401 A-150955 AUAGUUCAUUAACUCUUUU 6910-6928 2681 AD-75225 A-150956 AUGAGAAUUGGGAUUACAU 6927-6945 2402 A-150957 AUGUAAUCCCAAUUCUCAU 6927-6945 2682 AD-75226 A-150958 AUCAUGUAUUUUGCCUCAU 6944-6962 2403 A-150959 AUGAGGCAAAAUACAUGAU 6944-6962 2683 AD-75227 A-150960 UUAUCACACUUAUAGGCCA 6969-6987 2404 A-150961 UGGCCUAUAAGUGUGAUAA 6969-6987 2684 AD-75228 A-150962 CAAGUGUGAUAAAUAAACU 6986-7004 2405 A-150963 AGUUUAUUUAUCACACUUG 6986-7004 2685 AD-75229 A-150964 UUACAGACACUGAAUUAAU 7004-7022 2406 A-150965 AUUAAUUCAGUGUCUGUAA 7004-7022 2686 AD-75230 A-150966 UUUGAAACCAGAAAAUAAU 7035-7053 2407 A-150967 AUUAUUUUCUGGUUUCAAA 7035-7053 2687 AD-75231 A-150968 AUGACUGGCCAUUCGUUAA 7052-7070 2408 A-150969 UUAACGAAUGGCCAGUCAU 7052-7070 2688 AD-75232 A-150970 UUAGUUGAAAAGCAUAUUU 7078-7096 2409 A-150971 AAAUAUGCUUUUCAACUAA 7078-7096 2689 AD-75233 A-150972 UUUUUAUUAAAUUAAUUCU 7095-7113 2410 A-150973 AGAAUUAAUUUAAUAAAAA 7095-7113 2690 AD-75234 A-150974 CUGAUUGUAUUUGAAAUUA 7112-7130 2411 A-150975 UAAUUUCAAAUACAAUCAG 7112-7130 2691 AD-75235 A-150976 UUUGAAAUUAUUAUUCAAU 7121-7139 2412 A-150977 AUUGAAUAAUAAUUUCAAA 7121-7139 2692 AD-75236 A-150978 UUAUGGCAGAGGAAUAUCA 7144-7162 2413 A-150979 UGAUAUUCCUCUGCCAUAA 7144-7162 2693 AD-75237 A-150980 UCUAAAAAUGUAACUAAUU 7175-7193 2414 A-150981 AAUUAGUUACAUUUUUAGA 7175-7193 2694 AD-75238 A-150982 UUUACUGUUUAAUAAGCAU 7210-7228 2415 A-150983 AUGCUUAUUAAACAGUAAA 7210-7228 2695 AD-75239 A-150984 UGUCAUAAUAAAAUGGUAU 7252-7270 2416 A-150985 AUACCAUUUUAUUAUGACA 7252-7270 2696 AD-75240 A-150986 AUAUCUUUCUUUAGUAAUU 7269-7287 2417 A-150987 AAUUACUAAAGAAAGAUAU 7269-7287 2697 AD-75241 A-150988 UUAGUAAUUACAUUAAAAU 7279-7297 2418 A-150989 AUUUUAAUGUAAUUACUAA 7279-7297 2698 AD-75242 A-150990 AUUAGUCAUGUUUGAUUAA 7296-7314 2419 A-150991 UUAAUCAAACAUGACUAAU 7296-7314 2699

TABLE 19 IGF-1 in vitro 10 nM screen Duplex 10 nM 10 nM Position in Name AVG STD NM_000618.3 AD-74963 2.44 2.3  6-24 AD-74964 7.2 5.49 24-42 AD-74965 3.3 3.72 41-59 AD-74966 4.25 1.91 54-72 AD-74967 15.72 4.3 72-90 AD-74968 3.11 0.38 127-145 AD-74969 17.28 0.98 185-203 AD-74970 7.75 1.22 203-221 AD-74971 4.93 4 220-238 AD-74972 21.83 3.83 247-265 AD-74973 14.71 5.65 277-295 AD-74974 38.48 1.73 430-448 AD-74975 8.99 1.93 447-465 AD-74976 22.76 2.57 462-480 AD-74977 34.47 2.7 543-561 AD-74978 10.33 10.14 654-672 AD-74979 4.03 0.6 672-690 AD-74980 22.84 18.43 750-768 AD-74981 10.09 7.19 774-792 AD-74982 5.27 0.53 792-810 AD-74983 7.33 3.35 818-836 AD-74984 25.15 1.05 835-853 AD-74985 9.51 1.12 852-870 AD-74986 13.08 1.24 894-912 AD-74987 15.81 0.07 912-930 AD-74988 74.25 9.8 930-948 AD-74989 53.17 16.23 947-965 AD-74990 34.92 1.88 1091-1109 AD-74991 35.6 2.75 1108-1126 AD-74992 54.21 4.47 1125-1143 AD-74993 51.57 3.65 1135-1153 AD-74994 20.06 0.5 1144-1162 AD-74995 49.73 0.85 1162-1180 AD-74996 54.67 2.95 1195-1213 AD-74997 24.76 9.85 1197-1215 AD-74998 116.31 7.45 1215-1233 AD-74999 37.97 1.63 1232-1250 AD-75000 17.29 1.27 1293-1311 AD-75001 44.75 3.5 1311-1329 AD-75002 33.61 1.4 1334-1352 AD-75003 50.72 3.07 1352-1370 AD-75004 49.47 3.37 1370-1388 AD-75005 33.73 0.68 1388-1406 AD-75006 62.64 3.34 1406-1424 AD-75007 36.36 0.24 1423-1441 AD-75008 37.81 2.85 1440-1458 AD-75009 19.35 1.31 1472-1490 AD-75010 71.78 3.37 1494-1512 AD-75011 25.82 2.55 1511-1529 AD-75012 55.66 5.16 1528-1546 AD-75013 83.97 4.37 1572-1590 AD-75014 29.26 11.24 1599-1617 AD-75015 40.19 0.14 1625-1643 AD-75016 38.98 4.61 1643-1661 AD-75017 32.96 4.54 1690-1708 AD-75018 19.3 1.74 1709-1727 AD-75019 71.36 5.08 1757-1775 AD-75020 20.46 1.94 1793-1811 AD-75021 33.72 1.42 1807-1825 AD-75022 84.23 3.34 1825-1843 AD-75023 24.26 1.03 1843-1861 AD-75024 40.83 2.06 1966-1984 AD-75025 48.65 1.28 2016-2034 AD-75026 70.35 5.83 2033-2051 AD-75027 94.74 14.09 2040-2058 AD-75028 29.93 7.99 2057-2075 AD-75029 31.95 1.57 2090-2108 AD-75030 64.72 8.6 2140-2158 AD-75031 44.8 3.93 2170-2188 AD-75032 32.33 2.6 2192-2210 AD-75033 26.13 0.64 2210-2228 AD-75034 43.7 7.02 2228-2246 AD-75035 70.89 7.82 2249-2267 AD-75036 106.4 4.66 2266-2284 AD-75037 47.27 4.85 2285-2303 AD-75038 48.86 4.15 2303-2321 AD-75039 40.63 4.83 2322-2340 AD-75040 107.1 7.22 2340-2358 AD-75041 35.81 5.21 2357-2375 AD-75042 52.59 6.79 2398-2416 AD-75043 55.9 9.3 2432-2450 AD-75044 46.66 11.14 2449-2467 AD-75045 82.13 13.7 2453-2471 AD-75046 73.55 5.15 2470-2488 AD-75047 71.67 15.96 2482-2500 AD-75048 38.18 30.65 2521-2539 AD-75049 92.97 13.14 2539-2557 AD-75050 86 15.13 2548-2566 AD-75051 59.23 8.69 2566-2584 AD-75052 104.39 19.17 2584-2602 AD-75053 58.66 10.73 2601-2619 AD-75054 89.3 16.07 2618-2636 AD-75055 64.45 10.53 2623-2641 AD-75056 95.7 23.97 2640-2658 AD-75057 38.59 5.32 2648-2666 AD-75058 35.56 5.16 2666-2684 AD-75059 62.95 9.3 2684-2702 AD-75060 50.41 11.52 2771-2789 AD-75061 34.92 9.92 2793-2811 AD-75062 52.01 10.49 2811-2829 AD-75063 49.98 9.93 2828-2846 AD-75064 113.1 26.07 2869-2887 AD-75065 73.65 7.55 2906-2924 AD-75066 43.56 5.6 2920-2938 AD-75067 56.32 9.44 2938-2956 AD-75068 57.61 8.64 2956-2974 AD-75069 34.69 12.05 2962-2980 AD-75070 89.29 14.15 2980-2998 AD-75071 64.02 16.69 3006-3024 AD-75072 125.6 49.27 3025-3043 AD-75073 111.64 19.51 3043-3061 AD-75074 75.61 13.18 3060-3078 AD-75075 111.51 16.74 3077-3095 AD-75076 69.43 9.4 3100-3118 AD-75077 44.04 5 3137-3155 AD-75078 57.27 10.2 3155-3173 AD-75079 28.28 5.64 3208-3226 AD-75080 59.53 7.5 3225-3243 AD-75081 61.41 5.87 3234-3252 AD-75082 54.31 7.14 3252-3270 AD-75083 34.99 5.51 3269-3287 AD-75084 46.86 9.9 3277-3295 AD-75085 56.82 7.96 3294-3312 AD-75086 58.83 11.06 3334-3352 AD-75087 55.26 2.93 3352-3370 AD-75088 34.12 16.28 3369-3387 AD-75089 63.74 11.09 3400-3418 AD-75090 36.81 7.73 3417-3435 AD-75091 31.56 5.56 3449-3467 AD-75092 61.39 11.47 3517-3535 AD-75093 83.2 19.37 3535-3553 AD-75094 80.16 13.64 3567-3585 AD-75095 38.33 8.26 3585-3603 AD-75096 103.57 22.84 3603-3621 AD-75097 69.98 7.03 3621-3639 AD-75098 51.57 14.6 3662-3680 AD-75099 31.97 13.18 3686-3704 AD-75100 94.94 9.26 3704-3722 AD-75101 36.43 11.54 3721-3739 AD-75102 70.66 7.75 3732-3750 AD-75103 57.38 5.65 3750-3768 AD-75104 77.58 15.39 3767-3785 AD-75105 70.13 8.3 3770-3788 AD-75106 50.24 6.25 3788-3806 AD-75107 34.8 6.73 3806-3824 AD-75108 58.82 3.73 3835-3853 AD-75109 65.08 10.73 3865-3883 AD-75110 31.63 14.97 3939-3957 AD-75111 5.82 0.91 3982-4000 AD-75112 11.18 0.76 4000-4018 AD-75113 38.66 8.55 4081-4099 AD-75114 14.58 5.96 4098-4116 AD-75115 12.98 2.49 4101-4119 AD-75116 35.3 6.1 4119-4137 AD-75117 57.1 9.56 4137-4155 AD-75118 23.23 4.43 4154-4172 AD-75119 54.12 7.09 4208-4226 AD-75120 15.15 2.22 4271-4289 AD-75121 19.41 4.81 4289-4307 AD-75122 33.51 8.79 4319-4337 AD-75123 10.61 1.98 4336-4354 AD-75124 22.01 6.31 4341-4359 AD-75125 10.88 0.88 4359-4377 AD-75126 84.91 10.8 4377-4395 AD-75127 71.33 14.6 4395-4413 AD-75128 78.44 5.21 4412-4430 AD-75129 76.77 25.96 4426-4444 AD-75130 15.56 6.25 4443-4461 AD-75131 13.16 4.18 4460-4478 AD-75132 86.74 17.91 4464-4482 AD-75133 36.15 4.26 4484-4502 AD-75134 51.96 9.57 4511-4529 AD-75135 19.14 4.52 4566-4584 AD-75136 43.64 6.37 4584-4602 AD-75137 3.8 0.43 4601-4619 AD-75138 40.31 6.39 4625-4643 AD-75139 20.1 4.15 4642-4660 AD-75140 53.32 1.96 4645-4663 AD-75141 51.87 3.72 4669-4687 AD-75142 32.17 4.32 4686-4704 AD-75143 17.5 6.52 4699-4717 AD-75144 94.26 24.89 4717-4735 AD-75145 8.73 1.94 4734-4752 AD-75146 39.8 9.22 4751-4769 AD-75147 44.81 9.36 4770-4788 AD-75148 21.99 3.67 4780-4798 AD-75149 56.19 8.99 4798-4816 AD-75150 22.7 2.67 4815-4833 AD-75151 33.03 2.82 4825-4843 AD-75152 28.29 11.66 4884-4902 AD-75153 34.14 6.63 4911-4929 AD-75154 43.03 6.81 4987-5005 AD-75155 10.11 2.68 5004-5022 AD-75156 49.77 4.33 5016-5034 AD-75157 10.55 1.54 5050-5068 AD-75158 98.46 15.78 5098-5116 AD-75159 59.88 7.14 5115-5133 AD-75160 67.78 10.53 5127-5145 AD-75161 71.58 12.59 5153-5171 AD-75162 9.5 2.65 5171-5189 AD-75163 99.59 10.77 5189-5207 AD-75164 24.32 6.23 5206-5224 AD-75165 48.65 10.6 5223-5241 AD-75166 20.31 3.83 5238-5256 AD-75167 96.76 8.31 5284-5302 AD-75168 50.33 6.42 5301-5319 AD-75169 35.41 3.61 5311-5329 AD-75170 9.16 1.79 5328-5346 AD-75171 74.55 6.92 5383-5401 AD-75172 61.38 10.44 5391-5409 AD-75173 18.61 2.81 5409-5427 AD-75174 12.46 3.74 5412-5430 AD-75175 64.04 8.99 5430-5448 AD-75176 47.5 11.54 5505-5523 AD-75177 26.63 1.26 5570-5588 AD-75178 93.5 13.15 5603-5621 AD-75179 13.82 1.72 5620-5638 AD-75180 40.37 3.65 5628-5646 AD-75181 68.1 14 5645-5663 AD-75182 91.45 7.76 5659-5677 AD-75183 52.7 9.56 5677-5695 AD-75184 10.64 1.91 5694-5712 AD-75185 22.43 3.75 5715-5733 AD-75186 39.19 4.23 5740-5758 AD-75187 80.34 23.05 5773-5791 AD-75188 18.72 4.87 5790-5808 AD-75189 71.77 15.33 5884-5902 AD-75190 69.3 7.75 5917-5935 AD-75191 13.99 6.28 5934-5952 AD-75192 83.17 9.31 5942-5960 AD-75193 79.66 18.37 5959-5977 AD-75194 64.7 6.93 6066-6084 AD-75195 79.21 5.63 6129-6147 AD-75196 105.5 9.43 6147-6165 AD-75197 128.21 12.85 6164-6182 AD-75198 46.08 7.51 6231-6249 AD-75199 117.2 7.51 6303-6321 AD-75200 50.47 12.5 6346-6364 AD-75201 59.91 14.09 6364-6382 AD-75202 107.53 22.57 6382-6400 AD-75203 25.97 4.96 6399-6417 AD-75204 71.96 3.58 6444-6462 AD-75205 39.19 13.83 6461-6479 AD-75206 11.68 4.11 6479-6497 AD-75207 40.79 10.66 6497-6515 AD-75208 34.43 5.15 6517-6535 AD-75209 107.98 25.75 6538-6556 AD-75210 52.4 6.95 6557-6575 AD-75211 90.01 19.17 6576-6594 AD-75212 22.91 6.75 6594-6612 AD-75213 87.12 9.51 6612-6630 AD-75214 19.83 5.95 6629-6647 AD-75215 50.88 6.82 6653-6671 AD-75216 43.88 4.7 6722-6740 AD-75217 18.19 2.83 6762-6780 AD-75218 9.91 1.3 6806-6824 AD-75219 54.72 5.78 6824-6842 AD-75220 60.73 10.07 6841-6859 AD-75221 23.15 1.19 6858-6876 AD-75222 36.29 4.92 6870-6888 AD-75223 21.88 2.24 6887-6905 AD-75224 32.13 3.75 6910-6928 AD-75225 12.65 3.49 6927-6945 AD-75226 48.19 14.5 6944-6962 AD-75227 58.51 8.21 6969-6987 AD-75228 53.16 6.09 6986-7004 AD-75229 10.94 2.92 7004-7022 AD-75230 31.83 5.18 7035-7053 AD-75231 15.75 2.98 7052-7070 AD-75232 18.71 3.47 7078-7096 AD-75233 106.56 8.46 7095-7113 AD-75234 18.49 4.54 7112-7130 AD-75235 113.68 22.34 7121-7139 AD-75236 20.92 10.52 7144-7162 AD-75237 29.1 4.1 7175-7193 AD-75238 39.59 10.16 7210-7228 AD-75239 23.08 3.81 7252-7270 AD-75240 98.59 16.79 7269-7287 AD-75241 109.49 8.89 7279-7297 AD-75242 92.6 7.78 7296-7314

TABLE 20 Modified Sense and Antisense Strand Sequences of IGF-1 dsRNAs Sense SEQ Antisense  SEQ SEQ Duplex Oligo ID Oligo ID ID Name Name Sense Sequence NO Name Antisense Sequence NO mRNA target sequence NO AD-74963 A-150432 UAGAUAAAUGUGAGGAUUUdTdT 2700 A-150433 AAAUCCUCACAUUUAUCUAdTdT 2980 UAGAUAAAUGUGAGGAUUU 3260 AD-74964 A-150434 UUCUCUAAAUCCCUCUUCUdTdT 2701 A-150435 AGAAGAGGGAUUUAGAGAAdTdT 2981 UUCUCUAAAUCCCUCUUCU 3261 AD-74965 A-150436 CUGUUUGCUAAAUCUCACUdTdT 2702 A-150437 AGUGAGAUUUAGCAAACAGdTdT 2982 CUGUUUGCUAAAUCUCACU 3262 AD-74966 A-150438 CUCACUGUCACUGCUAAAUdTdT 2703 A-150439 AUUUAGCAGUGACAGUGAGdTdT 2983 CUCACUGUCACUGCUAAAU 3263 AD-74967 A-150440 UUCAGAGCAGAUAGAGCCUdTdT 2704 A-150441 AGGCUCUAUCUGCUCUGAAdTdT 2984 UUCAGAGCAGAUAGAGCCU 3264 AD-74968 A-150442 CAUUGCUCUCAACAUCUCAdTdT 2705 A-150443 UGAGAUGUUGAGAGCAAUGdTdT 2985 CAUUGCUCUCAACAUCUCC 3265 AD-74969 A-150444 ACCAAUUCAUUUUCAGACUdTdT 2706 A-150445 AGUCUGAAAAUGAAUUGGUdTdT 2986 ACCAAUUCAUUUUCAGACU 3266 AD-74970 A-150446 UUUGUACUUCAGAAGCAAUdTdT 2707 A-150447 AUUGCUUCUGAAGUACAAAdTdT 2987 UUUGUACUUCAGAAGCAAU 3267 AD-74971 A-150448 AUGGGAAAAAUCAGCAGUAdTdT 2708 A-150449 UACUGCUGAUUUUUCCCAUdTdT 2988 AUGGGAAAAAUCAGCAGUC 3268 AD-74972 A-150450 CAAUUAUUUAAGUGCUGCUdTdT 2709 A-150451 AGCAGCACUUAAAUAAUUGdTdT 2989 CAAUUAUUUAAGUGCUGCU 3269 AD-74973 A-150452 UUGAAGGUGAAGAUGCACAdTdT 2710 A-150453 UGUGCAUCUUCACCUUCAAdTdT 2990 UUGAAGGUGAAGAUGCACA 3270 AD-74974 A-150454 UUUUAUUUCAACAAGCCCAdTdT 2711 A-150455 UGGGCUUGUUGAAAUAAAAdTdT 2991 UUUUAUUUCAACAAGCCCA 3271 AD-74975 A-150456 CACAGGGUAUGGCUCCAGAdTdT 2712 A-150457 UCUGGAGCCAUACCCUGUGdTdT 2992 CACAGGGUAUGGCUCCAGC 3272 AD-74976 A-150458 CAGCAGUCGGAGGGCGCCUdTdT 2713 A-150459 AGGCGCCCUCCGACUGCUGdTdT 2993 CAGCAGUCGGAGGGCGCCU 3273 AD-74977 A-150460 UUGCGCACCCCUCAAGCCUdTdT 2714 A-150461 AGGCUUGAGGGGUGCGCAAdTdT 2994 UUGCGCACCCCUCAAGCCU 3274 AD-74978 A-150462 UGCAGGAAACAAGAACUAAdTdT 2715 A-150463 UUAGUUCUUGUUUCCUGCAdTdT 2995 UGCAGGAAACAAGAACUAC 3275 AD-74979 A-150464 CAGGAUGUAGGAAGACCCUdTdT 2716 A-150465 AGGGUCUUCCUACAUCCUGdTdT 2996 CAGGAUGUAGGAAGACCCU 3276 AD-74980 A-150466 UUAAACUUUGGAACACCUAdTdT 2717 A-150467 UAGGUGUUCCAAAGUUUAAdTdT 2997 UUAAACUUUGGAACACCUA 3277 AD-74981 A-150468 AAAUAAGUUUGAUAACAUUdTdT 2718 A-150469 AAUGUUAUCAAACUUAUUUdTdT 2998 AAAUAAGUUUGAUAACAUU 3278 AD-74982 A-150470 UUAAAAGAUGGGCGUUUCAdTdT 2719 A-150471 UGAAACGCCCAUCUUUUAAdTdT 2999 UUAAAAGAUGGGCGUUUCC 3279 AD-74983 A-150472 AAAUACACAAGUAAACAUUdTdT 2720 A-150473 AAUGUUUACUUGUGUAUUUdTdT 3000 AAAUACACAAGUAAACAUU 3280 AD-74984 A-150474 UUCCAACAUUGUCUUUAGAdTdT 2721 A-150475 UCUAAAGACAAUGUUGGAAdTdT 3001 UUCCAACAUUGUCUUUAGG 3281 AD-74985 A-150476 GGAGUGAUUUGCACCUUGAdTdT 2722 A-150477 UCAAGGUGCAAAUCACUCCdTdT 3002 GGAGUGAUUUGCACCUUGC 3282 AD-74986 A-150478 AUUGCUGUUGAUCUUUUAUdTdT 2723 A-150479 AUAAAAGAUCAACAGCAAUdTdT 3003 AUUGCUGUUGAUCUUUUAU 3283 AD-74987 A-150480 UCAAUAAUGUUCUAUAGAAdTdT 2724 A-150481 UUCUAUAGAACAUUAUUGAdTdT 3004 UCAAUAAUGUUCUAUAGAA 3284 AD-74988 A-150482 AAAGAAAAAAAAAAUAUAUdTdT 2725 A-150483 AUAUAUUUUUUUUUUCUUUdTdT 3005 AAAGAAAAAAAAAAUAUAU 3285 AD-74989 A-150484 AUAUAUAUAUAUAUCUUAAdTdT 2726 A-150485 UUAAGAUAUAUAUAUAUAUdTdT 3006 AUAUAUAUAUAUAUCUUAG 3286 AD-74990 A-150486 UUUCCUUAUUUGCACUUCUdTdT 2727 A-150487 AGAAGUGCAAAUAAGGAAAdTdT 3007 UUUCCUUAUUUGCACUUCU 3287 AD-74991 A-150488 CUUUCUACACAACUCGGGAdTdT 2728 A-150489 UCCCGAGUUGUGUAGAAAGdTdT 3008 CUUUCUACACAACUCGGGC 3288 AD-74992 A-150490 GCUGUUUGUUUUACAGUGUdTdT 2729 A-150491 ACACUGUAAAACAAACAGCdTdT 3009 GCUGUUUGUUUUACAGUGU 3289 AD-74993 A-150492 UUACAGUGUCUGAUAAUCUdTdT 2730 A-150493 AGAUUAUCAGACACUGUAAdTdT 3010 UUACAGUGUCUGAUAAUCU 3290 AD-74994 A-150494 CUGAUAAUCUUGUUAGUCUdTdT 2731 A-150495 AGACUAACAAGAUUAUCAGdTdT 3011 CUGAUAAUCUUGUUAGUCU 3291 AD-74995 A-150496 UAUACCCACCACCUCCCUUdTdT 2732 A-150497 AAGGGAGGUGGUGGGUAUAdTdT 3012 UAUACCCACCACCUCCCUU 3292 AD-74996 A-150498 UUGCCGAAUUUGGCCUCCUdTdT 2733 A-150499 AGGAGGCCAAAUUCGGCAAdTdT 3013 UUGCCGAAUUUGGCCUCCU 3293 AD-74997 A-150500 GCCGAAUUUGGCCUCCUCAdTdT 2734 A-150501 UGAGGAGGCCAAAUUCGGCdTdT 3014 GCCGAAUUUGGCCUCCUCA 3294 AD-74998 A-150502 AAAAGCAGCAGCAAGUCGUdTdT 2735 A-150503 ACGACUUGCUGCUGCUUUUdTdT 3015 AAAAGCAGCAGCAAGUCGU 3295 AD-74999 A-150504 GUCAAGAAGCACACCAAUUdTdT 2736 A-150505 AAUUGGUGUGCUUCUUGACdTdT 3016 GUCAAGAAGCACACCAAUU 3296 AD-75000 A-150506 AGUUGGAUGCAUUUUAUUUdTdT 2737 A-150507 AAAUAAAAUGCAUCCAACUdTdT 3017 AGUUGGAUGCAUUUUAUUU 3297 AD-75001 A-150508 UUAGACACAAAGCUUUAUUdTdT 2738 A-150509 AAUAAAGCUUUGUGUCUAAdTdT 3018 UUAGACACAAAGCUUUAUU 3298 AD-75002 A-150510 CACAUCAUGCUUACAAAAAdTdT 2739 A-150511 UUUUUGUAAGCAUGAUGUGdTdT 3019 CACAUCAUGCUUACAAAAA 3299 AD-75003 A-150512 AAGAAUAAUGCAAAUAGUUdTdT 2740 A-150513 AACUAUUUGCAUUAUUCUUdTdT 3020 AAGAAUAAUGCAAAUAGUU 3300 AD-75004 A-150514 UGCAACUUUGAGGCCAAUAdTdT 2741 A-150515 UAUUGGCCUCAAAGUUGCAdTdT 3021 UGCAACUUUGAGGCCAAUC 3301 AD-75005 A-150516 CAUUUUUAGGCAUAUGUUUdTdT 2742 A-150517 AAACAUAUGCCUAAAAAUGdTdT 3022 CAUUUUUAGGCAUAUGUUU 3302 AD-75006 A-150518 UUAAACAUAGAAAGUUUCUdTdT 2743 A-150519 AGAAACUUUCUAUGUUUAAdTdT 3023 UUAAACAUAGAAAGUUUCU 3303 AD-75007 A-150520 CUUCAACUCAAAAGAGUUAdTdT 2744 A-150521 UAACUCUUUUGAGUUGAAGdTdT 3024 CUUCAACUCAAAAGAGUUC 3304 AD-75008 A-150522 UCCUUCAAAUGAUGAGUUAdTdT 2745 A-150523 UAACUCAUCAUUUGAAGGAdTdT 3025 UCCUUCAAAUGAUGAGUUA 3305 AD-75009 A-150524 UUAGUAACUUUCCUCUUUUdTdT 2746 A-150525 AAAAGAGGAAAGUUACUAAdTdT 3026 UUAGUAACUUUCCUCUUUU 3306 AD-75010 A-150526 UUUUUCCAUAUAGAGCACUdTdT 2747 A-150527 AGUGCUCUAUAUGGAAAAAdTdT 3027 UUUUUCCAUAUAGAGCACU 3307 AD-75011 A-150528 CUAUGUAAAUUUAGCAUAUdTdT 2748 A-150529 AUAUGCUAAAUUUACAUAGdTdT 3028 CUAUGUAAAUUUAGCAUAU 3308 AD-75012 A-150530 AUCAAUUAUACAGGAUAUAdTdT 2749 A-150531 UAUAUCCUGUAUAAUUGAUdTdT 3029 AUCAAUUAUACAGGAUAUA 3309 AD-75013 A-150532 UUUAGUAUAAUGGUGCUAUdTdT 2750 A-150533 AUAGCACCAUUAUACUAAAdTdT 3030 UUUAGUAUAAUGGUGCUAU 3310 AD-75014 A-150534 UUGUUAUAUGAAAGAGUCUdTdT 2751 A-150535 AGACUCUUUCAUAUAACAAdTdT 3031 UUGUUAUAUGAAAGAGUCU 3311 AD-75015 A-150536 ACGGUAAUACGUGAAAGCAdTdT 2752 A-150537 UGCUUUCACGUAUUACCGUdTdT 3032 ACGGUAAUACGUGAAAGCA 3312 AD-75016 A-150538 AAAACAAUAGGGGAAGCCUdTdT 2753 A-150539 AGGCUUCCCCUAUUGUUUUdTdT 3033 AAAACAAUAGGGGAAGCCU 3313 AD-75017 A-150540 UACUGAAAACACCAUCCAUdTdT 2754 A-150541 AUGGAUGGUGUUUUCAGUAdTdT 3034 UACUGAAAACACCAUCCAU 3314 AD-75018 A-150542 UUGGGAAAGAAGGCAAAGUdTdT 2755 A-150543 ACUUUGCCUUCUUUCCCAAdTdT 3035 UUGGGAAAGAAGGCAAAGU 3315 AD-75019 A-150544 UCAGACACAAAAGUCCACUdTdT 2756 A-150545 AGUGGACUUUUGUGUCUGAdTdT 3036 UCAGACACAAAAGUCCACU 3316 AD-75020 A-150546 CGAGUCCAGAGAGGAAACUdTdT 2757 A-150547 AGUUUCCUCUCUGGACUCGdTdT 3037 CGAGUCCAGAGAGGAAACU 3317 AD-75021 A-150548 AAACUGUGGAAUGGAAAAAdTdT 2758 A-150549 UUUUUCCAUUCCACAGUUUdTdT 3038 AAACUGUGGAAUGGAAAAA 3318 AD-75022 A-150550 AGCAGAAGGCUAGGAAUUUdTdT 2759 A-150551 AAAUUCCUAGCCUUCUGCUdTdT 3039 AGCAGAAGGCUAGGAAUUU 3319 AD-75023 A-150552 UUAGCAGUCCUGGUUUCUUdTdT 2760 A-150553 AAGAAACCAGGACUGCUAAdTdT 3040 UUAGCAGUCCUGGUUUCUU 3320 AD-75024 A-150554 CAAAAUGGGGGCAAUAUGUdTdT 2761 A-150555 ACAUAUUGCCCCCAUUUUGdTdT 3041 CAAAAUGGGGGCAAUAUGU 3321 AD-75025 A-150556 UUUAAAAAGAUAAAGAUUAdTdT 2762 A-150557 UAAUCUUUAUCUUUUUAAAdTdT 3042 UUUAAAAAGAUAAAGAUUC 3322 AD-75026 A-150558 UCAGAUUUUUUUUACCCUAdTdT 2763 A-150559 UAGGGUAAAAAAAAUCUGAdTdT 3043 UCAGAUUUUUUUUACCCUG 3323 AD-75027 A-150560 UUUUUUACCCUGGGUUGCUdTdT 2764 A-150561 AGCAACCCAGGGUAAAAAAdTdT 3044 UUUUUUACCCUGGGUUGCU 3324 AD-75028 A-150562 CUGUAAGGGUGCAACAUCAdTdT 2765 A-150563 UGAUGUUGCACCCUUACAGdTdT 3045 CUGUAAGGGUGCAACAUCA 3325 AD-75029 A-150564 CUGAGAUGCAAGGAAUUCUdTdT 2766 A-150565 AGAAUUCCUUGCAUCUCAGdTdT 3046 CUGAGAUGCAAGGAAUUCU 3326 AD-75030 A-150566 UUGGUGAAUUGAAUGCUCAdTdT 2767 A-150567 UGAGCAUUCAAUUCACCAAdTdT 3047 UUGGUGAAUUGAAUGCUCC 3327 AD-75031 A-150568 UUCUUGUCAGUGAAGCUAUdTdT 2768 A-150569 AUAGCUUCACUGACAAGAAdTdT 3048 UUCUUGUCAGUGAAGCUAU 3328 AD-75032 A-150570 AAUAACUGGCCAACUAGUUdTdT 2769 A-150571 AACUAGUUGGCCAGUUAUUdTdT 3049 AAUAACUGGCCAACUAGUU 3329 AD-75033 A-150572 UGUUAAAAGCUAACAGCUAdTdT 2770 A-150573 UAGCUGUUAGCUUUUAACAdTdT 3050 UGUUAAAAGCUAACAGCUC 3330 AD-75034 A-150574 CAAUCUCUUAAAACACUUUdTdT 2771 A-150575 AAAGUGUUUUAAGAGAUUGdTdT 3051 CAAUCUCUUAAAACACUUU 3331 AD-75035 A-150576 AAAAUAUGUGGGAAGCAUUdTdT 2772 A-150577 AAUGCUUCCCACAUAUUUUdTdT 3052 AAAAUAUGUGGGAAGCAUU 3332 AD-75036 A-150578 UUUGAUUUUCAAUUUGAUUdTdT 2773 A-150579 AAUCAAAUUGAAAAUCAAAdTdT 3053 UUUGAUUUUCAAUUUGAUU 3333 AD-75037 A-150580 UUGAAUUCUGCAUUUGGUUdTdT 2774 A-150581 AACCAAAUGCAGAAUUCAAdTdT 3054 UUGAAUUCUGCAUUUGGUU 3334 AD-75038 A-150582 UUUAUGAAUACAAAGAUAAdTdT 2775 A-150583 UUAUCUUUGUAUUCAUAAAdTdT 3055 UUUAUGAAUACAAAGAUAA 3335 AD-75039 A-150584 GUGAAAAGAGAGAAAGGAAdTdT 2776 A-150585 UUCCUUUCUCUCUUUUCACdTdT 3056 GUGAAAAGAGAGAAAGGAA 3336 AD-75040 A-150586 AAAGAAAAAGGAGAAAAACdTdT 2777 A-150587 GUUUUUCUCCUUUUUCUUUdTdT 3057 AAAGAAAAAGGAGAAAAAC 3337 AD-75041 A-150588 ACAAAGAGAUUUCUACCAAdTdT 2778 A-150589 UUGGUAGAAAUCUCUUUGUdTdT 3058 ACAAAGAGAUUUCUACCAG 3338 AD-75042 A-150590 UUGUUAGCACUCACUGACUdTdT 2779 A-150591 AGUCAGUGAGUGCUAACAAdTdT 3059 UUGUUAGCACUCACUGACU 3339 AD-75043 A-150592 UACAUAUCUAGUAAAACCUdTdT 2780 A-150593 AGGUUUUACUAGAUAUGUAdTdT 3060 UACAUAUCUAGUAAAACCU 3340 AD-75044 A-150594 CUCGUUUAAUACUAUAAAUdTdT 2781 A-150595 AUUUAUAGUAUUAAACGAGdTdT 3061 CUCGUUUAAUACUAUAAAU 3341 AD-75045 A-150596 UUUAAUACUAUAAAUAAUAdTdT 2782 A-150597 UAUUAUUUAUAGUAUUAAAdTdT 3062 UUUAAUACUAUAAAUAAUA 3342 AD-75046 A-150598 UAUUCUAUUCAUUUUGAAAdTdT 2783 A-150599 UUUCAAAAUGAAUAGAAUAdTdT 3063 UAUUCUAUUCAUUUUGAAA 3343 AD-75047 A-150600 UUUGAAAAACACAAUGAUUdTdT 2784 A-150601 AAUCAUUGUGUUUUUCAAAdTdT 3064 UUUGAAAAACACAAUGAUU 3344 AD-75048 A-150602 AAGGAAAGUGAUCCAAAAUdTdT 2785 A-150603 AUUUUGGAUCACUUUCCUUdTdT 3065 AAGGAAAGUGAUCCAAAAU 3345 AD-75049 A-150604 UUUGAAAUAUUAAAAUAAUdTdT 2786 A-150605 AUUAUUUUAAUAUUUCAAAdTdT 3066 UUUGAAAUAUUAAAAUAAU 3346 AD-75050 A-150606 UUAAAAUAAUAUCUAAUAAdTdT 2787 A-150607 UUAUUAGAUAUUAUUUUAAdTdT 3067 UUAAAAUAAUAUCUAAUAA 3347 AD-75051 A-150608 AAAAGUCACAAAGUUAUCUdTdT 2788 A-150609 AGAUAACUUUGUGACUUUUdTdT 3068 AAAAGUCACAAAGUUAUCU 3348 AD-75052 A-150610 UUCUUUAACAAACUUUACUdTdT 2789 A-150611 AGUAAAGUUUGUUAAAGAAdTdT 3069 UUCUUUAACAAACUUUACU 3349 AD-75053 A-150612 CUCUUAUUCUUAGCUGUAUdTdT 2790 A-150613 AUACAGCUAAGAAUAAGAGdTdT 3070 CUCUUAUUCUUAGCUGUAU 3350 AD-75054 A-150614 AUAUACAUUUUUUUAAAAGdTdT 2791 A-150615 CUUUUAAAAAAAUGUAUAUdTdT 3071 AUAUACAUUUUUUUAAAAG 3351 AD-75055 A-150616 CAUUUUUUUAAAAGUUUGUdTdT 2792 A-150617 ACAAACUUUUAAAAAAAUGdTdT 3072 CAUUUUUUUAAAAGUUUGU 3352 AD-75056 A-150618 GUUAAAAUAUGCUUGACUAdTdT 2793 A-150619 UAGUCAAGCAUAUUUUAACdTdT 3073 GUUAAAAUAUGCUUGACUA 3353 AD-75057 A-150620 AUGCUUGACUAGAGUUUCAdTdT 2794 A-150621 UGAAACUCUAGUCAAGCAUdTdT 3074 AUGCUUGACUAGAGUUUCC 3354 AD-75058 A-150622 CAGUUGAAAGGCAAAAACUdTdT 2795 A-150623 AGUUUUUGCCUUUCAACUGdTdT 3075 CAGUUGAAAGGCAAAAACU 3355 AD-75059 A-150624 UUCCAUCACAACAAGAAAUdTdT 2796 A-150625 AUUUCUUGUUGUGAUGGAAdTdT 3076 UUCCAUCACAACAAGAAAU 3356 AD-75060 A-150626 UUGGUAUCAAGAAAGUCCAdTdT 2797 A-150627 UGGACUUUCUUGAUACCAAdTdT 3077 UUGGUAUCAAGAAAGUCCA 3357 AD-75061 A-150628 GUUAGUGUACUAGUCCAUAdTdT 2798 A-150629 UAUGGACUAGUACACUAACdTdT 3078 GUUAGUGUACUAGUCCAUC 3358 AD-75062 A-150630 CAUAGCCUAGAAAAUGAUAdTdT 2799 A-150631 UAUCAUUUUCUAGGCUAUGdTdT 3079 CAUAGCCUAGAAAAUGAUC 3359 AD-75063 A-150632 UCCCUAUCUGCAGAUCAAAdTdT 2800 A-150633 UUUGAUCUGCAGAUAGGGAdTdT 3080 UCCCUAUCUGCAGAUCAAG 3360 AD-75064 A-150634 UUAUCCAGCAUUCAGAUCUdTdT 2801 A-150635 AGAUCUGAAUGCUGGAUAAdTdT 3081 UUAUCCAGCAUUCAGAUCU 3361 AD-75065 A-150636 UUUUUGGUUAAAAGUACCAdTdT 2802 A-150637 UGGUACUUUUAACCAAAAAdTdT 3082 UUUUUGGUUAAAAGUACCC 3362 AD-75066 A-150638 UACCCAGGCUUGAUUAUUUdTdT 2803 A-150639 AAAUAAUCAAGCCUGGGUAdTdT 3083 UACCCAGGCUUGAUUAUUU 3363 AD-75067 A-150640 UCAUGCAAAUUCUAUAUUUdTdT 2804 A-150641 AAAUAUAGAAUUUGCAUGAdTdT 3084 UCAUGCAAAUUCUAUAUUU 3364 AD-75068 A-150642 UUACAUUCUUGGAAAGUCUdTdT 2805 A-150643 AGACUUUCCAAGAAUGUAAdTdT 3085 UUACAUUCUUGGAAAGUCU 3365 AD-75069 A-150644 UCUUGGAAAGUCUAUAUGAdTdT 2806 A-150645 UCAUAUAGACUUUCCAAGAdTdT 3086 UCUUGGAAAGUCUAUAUGA 3366 AD-75070 A-150646 AAAAACAAAAAUAACAUCUdTdT 2807 A-150647 AGAUGUUAUUUUUGUUUUUdTdT 3087 AAAAACAAAAAUAACAUCU 3367 AD-75071 A-150648 UUCUCCCACUGGGUCACCUdTdT 2808 A-150649 AGGUGACCCAGUGGGAGAAdTdT 3088 UUCUCCCACUGGGUCACCU 3368 AD-75072 A-150650 CAAGGAUCAGAGGCCAGGAdTdT 2809 A-150651 UCCUGGCCUCUGAUCCUUGdTdT 3089 CAAGGAUCAGAGGCCAGGA 3369 AD-75073 A-150652 AAAAAAAAAAAAAAGACUAdTdT 2810 A-150653 UAGUCUUUUUUUUUUUUUUdTdT 3090 AAAAAAAAAAAAAAGACUC 3370 AD-75074 A-150654 UCCCUGGAUCUCUGAAUAUdTdT 2811 A-150655 AUAUUCAGAGAUCCAGGGAdTdT 3091 UCCCUGGAUCUCUGAAUAU 3371 AD-75075 A-150656 AUAUGCAAAAAGAAGGCCAdTdT 2812 A-150657 UGGCCUUCUUUUUGCAUAUdTdT 3092 AUAUGCAAAAAGAAGGCCC 3372 AD-75076 A-150658 UAGUGGAGCCAGCAAUCCUdTdT 2813 A-150659 AGGAUUGCUGGCUCCACUAdTdT 3093 UAGUGGAGCCAGCAAUCCU 3373 AD-75077 A-150660 UUAACUCUCAGUCCAACAUdTdT 2814 A-150661 AUGUUGGACUGAGAGUUAAdTdT 3094 UUAACUCUCAGUCCAACAU 3374 AD-75078 A-150662 UUAUUUGAAUUGAGCACCUdTdT 2815 A-150663 AGGUGCUCAAUUCAAAUAAdTdT 3095 UUAUUUGAAUUGAGCACCU 3375 AD-75079 A-150664 CAGAUGUAAAAGAAACUAUdTdT 2816 A-150665 AUAGUUUCUUUUACAUCUGdTdT 3096 CAGAUGUAAAAGAAACUAU 3376 AD-75080 A-150666 AUACAUCAUUUUUGCCCUAdTdT 2817 A-150667 UAGGGCAAAAAUGAUGUAUdTdT 3097 AUACAUCAUUUUUGCCCUC 3377 AD-75081 A-150668 UUUUGCCCUCUGCCUGUUUdTdT 2818 A-150669 AAACAGGCAGAGGGCAAAAdTdT 3098 UUUUGCCCUCUGCCUGUUU 3378 AD-75082 A-150670 UUCCAGACAUACAGGUUCUdTdT 2819 A-150671 AGAACCUGUAUGUCUGGAAdTdT 3099 UUCCAGACAUACAGGUUCU 3379 AD-75083 A-150672 CUGUGGAAUAAGAUACUGAdTdT 2820 A-150673 UCAGUAUCUUAUUCCACAGdTdT 3100 CUGUGGAAUAAGAUACUGG 3380 AD-75084 A-150674 UAAGAUACUGGACUCCUCUdTdT 2821 A-150675 AGAGGAGUCCAGUAUCUUAdTdT 3101 UAAGAUACUGGACUCCUCU 3381 AD-75085 A-150676 CUUCCCAAGAUGGCACUUAdTdT 2822 A-150677 UAAGUGCCAUCUUGGGAAGdTdT 3102 CUUCCCAAGAUGGCACUUC 3382 AD-75086 A-150678 GUGUACCUUUUAAAAUUAUdTdT 2823 A-150679 AUAAUUUUAAAAGGUACACdTdT 3103 GUGUACCUUUUAAAAUUAU 3383 AD-75087 A-150680 UUCCCUCUCAACAAAACUUdTdT 2824 A-150681 AAGUUUUGUUGAGAGGGAAdTdT 3104 UUCCCUCUCAACAAAACUU 3384 AD-75088 A-150682 UUUAUAGGCAGUCUUCUGAdTdT 2825 A-150683 UCAGAAGACUGCCUAUAAAdTdT 3105 UUUAUAGGCAGUCUUCUGC 3385 AD-75089 A-150684 UUUUCUGUCAUAGUUAGAUdTdT 2826 A-150685 AUCUAACUAUGACAGAAAAdTdT 3106 UUUUCUGUCAUAGUUAGAU 3386 AD-75090 A-150686 AUGUGAUAAUUCUAAGAGUdTdT 2827 A-150687 ACUCUUAGAAUUAUCACAUdTdT 3107 AUGUGAUAAUUCUAAGAGU 3387 AD-75091 A-150688 UUCCUUCACUUAAUUCUAUdTdT 2828 A-150689 AUAGAAUUAAGUGAAGGAAdTdT 3108 UUCCUUCACUUAAUUCUAU 3388 AD-75092 A-150690 AUUAUCUUUCUUAACUUUUdTdT 2829 A-150691 AAAAGUUAAGAAAGAUAAUdTdT 3109 AUUAUCUUUCUUAACUUUU 3389 AD-75093 A-150692 UUCCAACACAUAAUCCUCUdTdT 2830 A-150693 AGAGGAUUAUGUGUUGGAAdTdT 3110 UUCCAACACAUAAUCCUCU 3390 AD-75094 A-150694 AAAUAAAUUGAAAAUAACUdTdT 2831 A-150695 AGUUAUUUUCAAUUUAUUUdTdT 3111 AAAUAAAUUGAAAAUAACU 3391 AD-75095 A-150696 UCAUUAUACCAAUUCACUAdTdT 2832 A-150697 UAGUGAAUUGGUAUAAUGAdTdT 3112 UCAUUAUACCAAUUCACUA 3392 AD-75096 A-150698 AUUUUAUUUUUUAAUGAAUdTdT 2833 A-150699 AUUCAUUAAAAAAUAAAAUdTdT 3113 AUUUUAUUUUUUAAUGAAU 3393 AD-75097 A-150700 UUAAAACUAGAAAACAAAUdTdT 2834 A-150701 AUUUGUUUUCUAGUUUUAAdTdT 3114 UUAAAACUAGAAAACAAAU 3394 AD-75098 A-150702 UUGAUUACUAUAUACUACAdTdT 2835 A-150703 UGUAGUAUAUAGUAAUCAAdTdT 3115 UUGAUUACUAUAUACUACA 3395 AD-75099 A-150704 AUGACUCAGAUUUCAUAGAdTdT 2836 A-150705 UCUAUGAAAUCUGAGUCAUdTdT 3116 AUGACUCAGAUUUCAUAGA 3396 AD-75100 A-150706 AAAGGAGCAACCAAAAUGUdTdT 2837 A-150707 ACAUUUUGGUUGCUCCUUUdTdT 3117 AAAGGAGCAACCAAAAUGU 3397 AD-75101 A-150708 GUCACAACCCAAAACUUUAdTdT 2838 A-150709 UAAAGUUUUGGGUUGUGACdTdT 3118 GUCACAACCCAAAACUUUA 3398 AD-75102 A-150710 AAACUUUACAAGCUUUGCUdTdT 2839 A-150711 AGCAAAGCUUGUAAAGUUUdTdT 3119 AAACUUUACAAGCUUUGCU 3399 AD-75103 A-150712 UUCAGAAUUAGAUUGCUUUdTdT 2840 A-150713 AAAGCAAUCUAAUUCUGAAdTdT 3120 UUCAGAAUUAGAUUGCUUU 3400 AD-75104 A-150714 UUAUAAUUCUUGAAUGAGAdTdT 2841 A-150715 UCUCAUUCAAGAAUUAUAAdTdT 3121 UUAUAAUUCUUGAAUGAGG 3401 AD-75105 A-150716 UAAUUCUUGAAUGAGGCAAdTdT 2842 A-150717 UUGCCUCAUUCAAGAAUUAdTdT 3122 UAAUUCUUGAAUGAGGCAA 3402 AD-75106 A-150718 AUUUCAAGAUAUUUGUAAAdTdT 2843 A-150719 UUUACAAAUAUCUUGAAAUdTdT 3123 AUUUCAAGAUAUUUGUAAA 3403 AD-75107 A-150720 AAGAACAGUAAACAUUGGUdTdT 2844 A-150721 ACCAAUGUUUACUGUUCUUdTdT 3124 AAGAACAGUAAACAUUGGU 3404 AD-75108 A-150722 UUUCAACUCAUAGGCUUAUdTdT 2845 A-150723 AUAAGCCUAUGAGUUGAAAdTdT 3125 UUUCAACUCAUAGGCUUAU 3405 AD-75109 A-150724 UUGACCAUACUGGAUACUUdTdT 2846 A-150725 AAGUAUCCAGUAUGGUCAAdTdT 3126 UUGACCAUACUGGAUACUU 3406 AD-75110 A-150726 UUUAAGAUGAGGCAGUUCAdTdT 2847 A-150727 UGAACUGCCUCAUCUUAAAdTdT 3127 UUUAAGAUGAGGCAGUUCC 3407 AD-75111 A-150728 CAUCAGAAUCCACUCUUCUdTdT 2848 A-150729 AGAAGAGUGGAUUCUGAUGdTdT 3128 CAUCAGAAUCCACUCUUCU 3408 AD-75112 A-150730 UAGGGAUAUGAAAAUCUCUdTdT 2849 A-150731 AGAGAUUUUCAUAUCCCUAdTdT 3129 UAGGGAUAUGAAAAUCUCU 3409 AD-75113 A-150732 UUCACCCUAAGGAUCCAAUdTdT 2850 A-150733 AUUGGAUCCUUAGGGUGAAdTdT 3130 UUCACCCUAAGGAUCCAAU 3410 AD-75114 A-150734 AUGGAAUACUGAAAAGAAAdTdT 2851 A-150735 UUUCUUUUCAGUAUUCCAUdTdT 3131 AUGGAAUACUGAAAAGAAA 3411 AD-75115 A-150736 GAAUACUGAAAAGAAAUCAdTdT 2852 A-150737 UGAUUUCUUUUCAGUAUUCdTdT 3132 GAAUACUGAAAAGAAAUCA 3412 AD-75116 A-150738 ACUUCCUUGAAAAUUUUAUdTdT 2853 A-150739 AUAAAAUUUUCAAGGAAGUdTdT 3133 ACUUCCUUGAAAAUUUUAU 3413 AD-75117 A-150740 UUAAAAAACAAACAAACAAdTdT 2854 A-150741 UUGUUUGUUUGUUUUUUAAdTdT 3134 UUAAAAAACAAACAAACAA 3414 AD-75118 A-150742 AAACAAAAAGCCUGUCCAAdTdT 2855 A-150743 UUGGACAGGCUUUUUGUUUdTdT 3135 AAACAAAAAGCCUGUCCAC 3415 AD-75119 A-150744 UUUGUGUAGAUGAAACCAUdTdT 2856 A-150745 AUGGUUUCAUCUACACAAAdTdT 3136 UUUGUGUAGAUGAAACCAU 3416 AD-75120 A-150746 UUGGGAGAAGGCUUAGAAUdTdT 2857 A-150747 AUUCUAAGCCUUCUCCCAAdTdT 3137 UUGGGAGAAGGCUUAGAAU 3417 AD-75121 A-150748 UAAAAGAUGUAGCACAUUUdTdT 2858 A-150749 AAAUGUGCUACAUCUUUUAdTdT 3138 UAAAAGAUGUAGCACAUUU 3418 AD-75122 A-150750 UUAUUGUUUGGCCAGCUAUdTdT 2859 A-150751 AUAGCUGGCCAAACAAUAAdTdT 3139 UUAUUGUUUGGCCAGCUAU 3419 AD-75123 A-150752 AUGCCAAUGUGGUGCUAUUdTdT 2860 A-150753 AAUAGCACCACAUUGGCAUdTdT 3140 AUGCCAAUGUGGUGCUAUU 3420 AD-75124 A-150754 AAUGUGGUGCUAUUGUUUAdTdT 2861 A-150755 UAAACAAUAGCACCACAUUdTdT 3141 AAUGUGGUGCUAUUGUUUC 3421 AD-75125 A-150756 CUUUAAGAAAGUACUUGAAdTdT 2862 A-150757 UUCAAGUACUUUCUUAAAGdTdT 3142 CUUUAAGAAAGUACUUGAC 3422 AD-75126 A-150758 CUAAAAAAAAAAGAAAAAAdTdT 2863 A-150759 UUUUUUCUUUUUUUUUUAGdTdT 3143 CUAAAAAAAAAAGAAAAAA 3423 AD-75127 A-150760 AAGAAAAAAAAGAAAGCAUdTdT 2864 A-150761 AUGCUUUCUUUUUUUUCUUdTdT 3144 AAGAAAAAAAAGAAAGCAU 3424 AD-75128 A-150762 AUAGACAUAUUUUUUUAAAdTdT 2865 A-150763 UUUAAAAAAAUAUGUCUAUdTdT 3145 AUAGACAUAUUUUUUUAAA 3425 AD-75129 A-150764 UUAAAGUAUAAAAACAACAdTdT 2866 A-150765 UGUUGUUUUUAUACUUUAAdTdT 3146 UUAAAGUAUAAAAACAACA 3426 AD-75130 A-150766 CAAUUCUAUAGAUAGAUGAdTdT 2867 A-150767 UCAUCUAUCUAUAGAAUUGdTdT 3147 CAAUUCUAUAGAUAGAUGG 3427 AD-75131 A-150768 GGCUUAAUAAAAUAGCAUUdTdT 2868 A-150769 AAUGCUAUUUUAUUAAGCCdTdT 3148 GGCUUAAUAAAAUAGCAUU 3428 AD-75132 A-150770 UAAUAAAAUAGCAUUAGGUdTdT 2869 A-150771 ACCUAAUGCUAUUUUAUUAdTdT 3149 UAAUAAAAUAGCAUUAGGU 3429 AD-75133 A-150772 UAUCUAGCCACCACCACCUdTdT 2870 A-150773 AGGUGGUGGUGGCUAGAUAdTdT 3150 UAUCUAGCCACCACCACCU 3430 AD-75134 A-150774 UUUAUCACUCACAAGUAGUdTdT 2871 A-150775 ACUACUUGUGAGUGAUAAAdTdT 3151 UUUAUCACUCACAAGUAGU 3431 AD-75135 A-150776 GGCAGGAGUUGGAAAUUUUdTdT 2872 A-150777 AAAAUUUCCAACUCCUGCCdTdT 3152 GGCAGGAGUUGGAAAUUUU 3432 AD-75136 A-150778 UUUAAAGUUAGAAGGCUCAdTdT 2873 A-150779 UGAGCCUUCUAACUUUAAAdTdT 3153 UUUAAAGUUAGAAGGCUCC 3433 AD-75137 A-150780 CCAUUGUUUUGUUGGCUCUdTdT 2874 A-150781 AGAGCCAACAAAACAAUGGdTdT 3154 CCAUUGUUUUGUUGGCUCU 3434 AD-75138 A-150782 UUAGCAAAAUUAGCAAUAUdTdT 2875 A-150783 AUAUUGCUAAUUUUGCUAAdTdT 3155 UUAGCAAAAUUAGCAAUAU 3435 AD-75139 A-150784 AUAUUAUCCAAUCUUCUGAdTdT 2876 A-150785 UCAGAAGAUUGGAUAAUAUdTdT 3156 AUAUUAUCCAAUCUUCUGA 3436 AD-75140 A-150786 UUAUCCAAUCUUCUGAACUdTdT 2877 A-150787 AGUUCAGAAGAUUGGAUAAdTdT 3157 UUAUCCAAUCUUCUGAACU 3437 AD-75141 A-150788 AAGAGCAUGGAGAAUAAACdTdT 2878 A-150789 GUUUAUUCUCCAUGCUCUUdTdT 3158 AAGAGCAUGGAGAAUAAAC 3438 AD-75142 A-150790 ACGCGGGAAAAAAGAUCUUdTdT 2879 A-150791 AAGAUCUUUUUUCCCGCGUdTdT 3159 ACGCGGGAAAAAAGAUCUU 3439 AD-75143 A-150792 GAUCUUAUAGGCAAAUAGAdTdT 2880 A-150793 UCUAUUUGCCUAUAAGAUCdTdT 3160 GAUCUUAUAGGCAAAUAGA 3440 AD-75144 A-150794 AAGAAUUUAAAAGAUAAGUdTdT 2881 A-150795 ACUUAUCUUUUAAAUUCUUdTdT 3161 AAGAAUUUAAAAGAUAAGU 3441 AD-75145 A-150796 GUAAGUUCCUUAUUGAUUUdTdT 2882 A-150797 AAAUCAAUAAGGAACUUACdTdT 3162 GUAAGUUCCUUAUUGAUUU 3442 AD-75146 A-150798 UUUUGUGCACUCUGCUCUAdTdT 2883 A-150799 UAGAGCAGAGUGCACAAAAdTdT 3163 UUUUGUGCACUCUGCUCUA 3443 AD-75147 A-150800 AAACAGAUAUUCAGCAAGUdTdT 2884 A-150801 ACUUGCUGAAUAUCUGUUUdTdT 3164 AAACAGAUAUUCAGCAAGU 3444 AD-75148 A-150802 UCAGCAAGUGGAGAAAAUAdTdT 2885 A-150803 UAUUUUCUCCACUUGCUGAdTdT 3165 UCAGCAAGUGGAGAAAAUA 3445 AD-75149 A-150804 AAGAACAAAGAGAAAAAAUdTdT 2886 A-150805 AUUUUUUCUCUUUGUUCUUdTdT 3166 AAGAACAAAGAGAAAAAAU 3446 AD-75150 A-150806 AUACAUAGAUUUACCUGCAdTdT 2887 A-150807 UGCAGGUAAAUCUAUGUAUdTdT 3167 AUACAUAGAUUUACCUGCA 3447 AD-75151 A-150808 UUACCUGCAAAAAAUAGCUdTdT 2888 A-150809 AGCUAUUUUUUGCAGGUAAdTdT 3168 UUACCUGCAAAAAAUAGCU 3448 AD-75152 A-150810 UUUAUAGAAGACAUUCUCAdTdT 2889 A-150811 UGAGAAUGUCUUCUAUAAAdTdT 3169 UUUAUAGAAGACAUUCUCC 3449 AD-75153 A-150812 AGACAUCUCAAAGAGCAGUdTdT 2890 A-150813 ACUGCUCUUUGAGAUGUCUdTdT 3170 AGACAUCUCAAAGAGCAGU 3450 AD-75154 A-150814 UAUGAGAUGGGGGUUAUCUdTdT 2891 A-150815 AGAUAACCCCCAUCUCAUAdTdT 3171 UAUGAGAUGGGGGUUAUCU 3451 AD-75155 A-150816 CUACUGAUAAAGAAAGAAUdTdT 2892 A-150817 AUUCUUUCUUUAUCAGUAGdTdT 3172 CUACUGAUAAAGAAAGAAU 3452 AD-75156 A-150818 AAAGAAUUUAUGAGAAAUUdTdT 2893 A-150819 AAUUUCUCAUAAAUUCUUUdTdT 3173 AAAGAAUUUAUGAGAAAUU 3453 AD-75157 A-150820 UAACAAUCUGUGAAGAUUUdTdT 2894 A-150821 AAAUCUUCACAGAUUGUUAdTdT 3174 UAACAAUCUGUGAAGAUUU 3454 AD-75158 A-150822 UUUUACUUUAUACAGUCUUdTdT 2895 A-150823 AAGACUGUAUAAAGUAAAAdTdT 3175 UUUUACUUUAUACAGUCUU 3455 AD-75159 A-150824 UUUAUGAAUUUCUUAAUGUdTdT 2896 A-150825 ACAUUAAGAAAUUCAUAAAdTdT 3176 UUUAUGAAUUUCUUAAUGU 3456 AD-75160 A-150826 UUAAUGUUCAAAAUGACUUdTdT 2897 A-150827 AAGUCAUUUUGAACAUUAAdTdT 3177 UUAAUGUUCAAAAUGACUU 3457 AD-75161 A-150828 UUCUUCUUUUUUUAUAUCAdTdT 2898 A-150829 UGAUAUAAAAAAAGAAGAAdTdT 3178 UUCUUCUUUUUUUAUAUCA 3458 AD-75162 A-150830 AGAAUGAGGAAUAAUAAGUdTdT 2899 A-150831 ACUUAUUAUUCCUCAUUCUdTdT 3179 AGAAUGAGGAAUAAUAAGU 3459 AD-75163 A-150832 UUAAACCCACAUAGACUCUdTdT 2900 A-150833 AGAGUCUAUGUGGGUUUAAdTdT 3180 UUAAACCCACAUAGACUCU 3460 AD-75164 A-150834 CUUUAAAACUAUAGGCUAAdTdT 2901 A-150835 UUAGCCUAUAGUUUUAAAGdTdT 3181 CUUUAAAACUAUAGGCUAG 3461 AD-75165 A-150836 AGAUAGAAAUGUAUGUUUAdTdT 2902 A-150837 UAAACAUACAUUUCUAUCUdTdT 3182 AGAUAGAAAUGUAUGUUUG 3462 AD-75166 A-150838 UUUGACUUGUUGAAGCUAUdTdT 2903 A-150839 AUAGCUUCAACAAGUCAAAdTdT 3183 UUUGACUUGUUGAAGCUAU 3463 AD-75167 A-150840 UUUUUAAUCUUAAAAGAUUdTdT 2904 A-150841 AAUCUUUUAAGAUUAAAAAdTdT 3184 UUUUUAAUCUUAAAAGAUU 3464 AD-75168 A-150842 UUGUGCUAAUUUAUUAGAAdTdT 2905 A-150843 UUCUAAUAAAUUAGCACAAdTdT 3185 UUGUGCUAAUUUAUUAGAG 3465 AD-75169 A-150844 UUAUUAGAGCAGAACCUGUdTdT 2906 A-150845 ACAGGUUCUGCUCUAAUAAdTdT 3186 UUAUUAGAGCAGAACCUGU 3466 AD-75170 A-150846 GUUUGGCUCUCCUCAGAAAdTdT 2907 A-150847 UUUCUGAGGAGAGCCAAACdTdT 3187 GUUUGGCUCUCCUCAGAAG 3467 AD-75171 A-150848 CAAUAUUUUCAAAAGAUAAdTdT 2908 A-150849 UUAUCUUUUGAAAAUAUUGdTdT 3188 CAAUAUUUUCAAAAGAUAA 3468 AD-75172 A-150850 UCAAAAGAUAAAUCUGAUUdTdT 2909 A-150851 AAUCAGAUUUAUCUUUUGAdTdT 3189 UCAAAAGAUAAAUCUGAUU 3469 AD-75173 A-150852 UUAUGCAAUGGCAUCAUUUdTdT 2910 A-150853 AAAUGAUGCCAUUGCAUAAdTdT 3190 UUAUGCAAUGGCAUCAUUU 3470 AD-75174 A-150854 UGCAAUGGCAUCAUUUAUUdTdT 2911 A-150855 AAUAAAUGAUGCCAUUGCAdTdT 3191 UGCAAUGGCAUCAUUUAUU 3471 AD-75175 A-150856 UUUAAAACAGAAGAAUUGUdTdT 2912 A-150857 ACAAUUCUUCUGUUUUAAAdTdT 3192 UUUAAAACAGAAGAAUUGU 3472 AD-75176 A-150858 AACAACAAAAGGAAAAUGUdTdT 2913 A-150859 ACAUUUUCCUUUUGUUGUUdTdT 3193 AACAACAAAAGGAAAAUGU 3473 AD-75177 A-150860 UUAAUCCUGUAGUACAUAUdTdT 2914 A-150861 AUAUGUACUACAGGAUUAAdTdT 3194 UUAAUCCUGUAGUACAUAU 3474 AD-75178 A-150862 UUUAAUAUUUUAUAAGACAdTdT 2915 A-150863 UGUCUUAUAAAAUAUUAAAdTdT 3195 UUUAAUAUUUUAUAAGACC 3475 AD-75179 A-150864 CCUUCCUGUUAGGUAUUAAdTdT 2916 A-150865 UUAAUACCUAACAGGAAGGdTdT 3196 CCUUCCUGUUAGGUAUUAG 3476 AD-75180 A-150866 UUAGGUAUUAGAAAGUGAUdTdT 2917 A-150867 AUCACUUUCUAAUACCUAAdTdT 3197 UUAGGUAUUAGAAAGUGAU 3477 AD-75181 A-150868 AUACAUAGAUAUCUUUUUUdTdT 2918 A-150869 AAAAAAGAUAUCUAUGUAUdTdT 3198 AUACAUAGAUAUCUUUUUU 3478 AD-75182 A-150870 UUUUUGUGUAAUUUCUAUUdTdT 2919 A-150871 AAUAGAAAUUACACAAAAAdTdT 3199 UUUUUGUGUAAUUUCUAUU 3479 AD-75183 A-150872 UUAAAAAAGAGAGAAGACUdTdT 2920 A-150873 AGUCUUCUCUCUUUUUUAAdTdT 3200 UUAAAAAAGAGAGAAGACU 3480 AD-75184 A-150874 CUGUCAGAAGCUUUAAGUAdTdT 2921 A-150875 UACUUAAAGCUUCUGACAGdTdT 3201 CUGUCAGAAGCUUUAAGUG 3481 AD-75185 A-150876 UAUGGUACAGGAUAAAGAUdTdT 2922 A-150877 AUCUUUAUCCUGUACCAUAdTdT 3202 UAUGGUACAGGAUAAAGAU 3482 AD-75186 A-150878 UUAAAUAACCAAUUCCUAUdTdT 2923 A-150879 AUAGGAAUUGGUUAUUUAAdTdT 3203 UUAAAUAACCAAUUCCUAU 3483 AD-75187 A-150880 UUGUUUUUUAAAGAAACCUdTdT 2924 A-150881 AGGUUUCUUUAAAAAACAAdTdT 3204 UUGUUUUUUAAAGAAACCU 3484 AD-75188 A-150882 CUCUCACAGAUAAGACAGAdTdT 2925 A-150883 UCUGUCUUAUCUGUGAGAGdTdT 3205 CUCUCACAGAUAAGACAGA 3485 AD-75189 A-150884 CAGAAUUUUAUAGAGGGCUdTdT 2926 A-150885 AGCCCUCUAUAAAAUUCUGdTdT 3206 CAGAAUUUUAUAGAGGGCU 3486 AD-75190 A-150886 UCUAGAAUUAAAGGAACCUdTdT 2927 A-150887 AGGUUCCUUUAAUUCUAGAdTdT 3207 UCUAGAAUUAAAGGAACCU 3487 AD-75191 A-150888 CUCACUGAAAACAUAUAUUdTdT 2928 A-150889 AAUAUAUGUUUUCAGUGAGdTdT 3208 CUCACUGAAAACAUAUAUU 3488 AD-75192 A-150890 AAACAUAUAUUUCACGUGUdTdT 2929 A-150891 ACACGUGAAAUAUAUGUUUdTdT 3209 AAACAUAUAUUUCACGUGU 3489 AD-75193 A-150892 GUUCCCUCUUUUUUUUUUUdTdT 2930 A-150893 AAAAAAAAAAAGAGGGAACdTdT 3210 GUUCCCUCUUUUUUUUUUU 3490 AD-75194 A-150894 UUAAGCGAUUCUCCUGCCUdTdT 2931 A-150895 AGGCAGGAGAAUCGCUUAAdTdT 3211 UUAAGCGAUUCUCCUGCCU 3491 AD-75195 A-150896 CGGCUAAUUUUUUGGAUUUdTdT 2932 A-150897 AAAUCCAAAAAAUUAGCCGdTdT 3212 CGGCUAAUUUUUUGGAUUU 3492 AD-75196 A-150898 UUUAAUAGAGACGGGGUUUdTdT 2933 A-150899 AAACCCCGUCUCUAUUAAAdTdT 3213 UUUAAUAGAGACGGGGUUU 3493 AD-75197 A-150900 UUUACCAUGUUGGCCAGGUdTdT 2934 A-150901 ACCUGGCCAACAUGGUAAAdTdT 3214 UUUACCAUGUUGGCCAGGU 3494 AD-75198 A-150902 UUGCUGGGAUUACAGGCAUdTdT 2935 A-150903 AUGCCUGUAAUCCCAGCAAdTdT 3215 UUGCUGGGAUUACAGGCAU 3495 AD-75199 A-150904 UUAAACAUGAUCCUUCUCUdTdT 2936 A-150905 AGAGAAGGAUCAUGUUUAAdTdT 3216 UUAAACAUGAUCCUUCUCU 3496 AD-75200 A-150906 GGGGUCUUUCAAGGGGAAAdTdT 2937 A-150907 UUUCCCCUUGAAAGACCCCdTdT 3217 GGGGUCUUUCAAGGGGAAA 3497 AD-75201 A-150908 AAAAAUCCAAGCUUUUUUAdTdT 2938 A-150909 UAAAAAAGCUUGGAUUUUUdTdT 3218 AAAAAUCCAAGCUUUUUUA 3498 AD-75202 A-150910 AAAGUAAAAAAAAAAAAAGdTdT 2939 A-150911 CUUUUUUUUUUUUUACUUUdTdT 3219 AAAGUAAAAAAAAAAAAAG 3499 AD-75203 A-150912 AGAGAGGACACAAAACCAAdTdT 2940 A-150913 UUGGUUUUGUGUCCUCUCUdTdT 3220 AGAGAGGACACAAAACCAA 3500 AD-75204 A-150914 UUAAGAUGGAGACAGAGUUdTdT 2941 A-150915 AACUCUGUCUCCAUCUUAAdTdT 3221 UUAAGAUGGAGACAGAGUU 3501 AD-75205 A-150916 UUUCUCCUAAUAACCGGAAdTdT 2942 A-150917 UUCCGGUUAUUAGGAGAAAdTdT 3222 UUUCUCCUAAUAACCGGAG 3502 AD-75206 A-150918 GCUGAAUUACCUUUCACUUdTdT 2943 A-150919 AAGUGAAAGGUAAUUCAGCdTdT 3223 GCUGAAUUACCUUUCACUU 3503 AD-75207 A-150920 UUCAAAAACAUGACCUUCAdTdT 2944 A-150921 UGAAGGUCAUGUUUUUGAAdTdT 3224 UUCAAAAACAUGACCUUCC 3504 AD-75208 A-150922 CAAUCCUUAGAAUCUGCCUdTdT 2945 A-150923 AGGCAGAUUCUAAGGAUUGdTdT 3225 CAAUCCUUAGAAUCUGCCU 3505 AD-75209 A-150924 UUUUAUAUUACUGAGGCCUdTdT 2946 A-150925 AGGCCUCAGUAAUAUAAAAdTdT 3226 UUUUAUAUUACUGAGGCCU 3506 AD-75210 A-150926 AAAAGUAAACAUUACUCAUdTdT 2947 A-150927 AUGAGUAAUGUUUACUUUUdTdT 3227 AAAAGUAAACAUUACUCAU 3507 AD-75211 A-150928 UUUAUUUUGCCCAAAAUGAdTdT 2948 A-150929 UCAUUUUGGGCAAAAUAAAdTdT 3228 UUUAUUUUGCCCAAAAUGC 3508 AD-75212 A-150930 CACUGAUGUAAAGUAGGAAdTdT 2949 A-150931 UUCCUACUUUACAUCAGUGdTdT 3229 CACUGAUGUAAAGUAGGAA 3509 AD-75213 A-150932 AAAAUAAAAACAGAGCUCUdTdT 2950 A-150933 AGAGCUCUGUUUUUAUUUUdTdT 3230 AAAAUAAAAACAGAGCUCU 3510 AD-75214 A-150934 CUAAAAUCCCUUUCAAGCAdTdT 2951 A-150935 UGCUUGAAAGGGAUUUUAGdTdT 3231 CUAAAAUCCCUUUCAAGCC 3511 AD-75215 A-150936 UUGACCCCACUCACCAACUdTdT 2952 A-150937 AGUUGGUGAGUGGGGUCAAdTdT 3232 UUGACCCCACUCACCAACU 3512 AD-75216 A-150938 UUAUCUUGUACCCGCUGCUdTdT 2953 A-150939 AGCAGCGGGUACAAGAUAAdTdT 3233 UUAUCUUGUACCCGCUGCU 3513 AD-75217 A-150940 CUGAAACCUCAAGCUGUCUdTdT 2954 A-150941 AGACAGCUUGAGGUUUCAGdTdT 3234 CUGAAACCUCAAGCUGUCU 3514 AD-75218 A-150942 GUAUCAUGAAAAUGUCUAUdTdT 2955 A-150943 AUAGACAUUUUCAUGAUACdTdT 3235 GUAUCAUGAAAAUGUCUAU 3515 AD-75219 A-150944 UUCAAAAUAUCAAAACCUUdTdT 2956 A-150945 AAGGUUUUGAUAUUUUGAAdTdT 3236 UUCAAAAUAUCAAAACCUU 3516 AD-75220 A-150946 UUUCAAAUAUCACGCAGCUdTdT 2957 A-150947 AGCUGCGUGAUAUUUGAAAdTdT 3237 UUUCAAAUAUCACGCAGCU 3517 AD-75221 A-150948 CUUAUAUUCAGUUUACAUAdTdT 2958 A-150949 UAUGUAAACUGAAUAUAAGdTdT 3238 CUUAUAUUCAGUUUACAUA 3518 AD-75222 A-150950 UUACAUAAAGGCCCCAAAUdTdT 2959 A-150951 AUUUGGGGCCUUUAUGUAAdTdT 3239 UUACAUAAAGGCCCCAAAU 3519 AD-75223 A-150952 AUACCAUGUCAGAUCUUUUdTdT 2960 A-150953 AAAAGAUCUGACAUGGUAUdTdT 3240 AUACCAUGUCAGAUCUUUU 3520 AD-75224 A-150954 AAAAGAGUUAAUGAACUAUdTdT 2961 A-150955 AUAGUUCAUUAACUCUUUUdTdT 3241 AAAAGAGUUAAUGAACUAU 3521 AD-75225 A-150956 AUGAGAAUUGGGAUUACAUdTdT 2962 A-150957 AUGUAAUCCCAAUUCUCAUdTdT 3242 AUGAGAAUUGGGAUUACAU 3522 AD-75226 A-150958 AUCAUGUAUUUUGCCUCAUdTdT 2963 A-150959 AUGAGGCAAAAUACAUGAUdTdT 3243 AUCAUGUAUUUUGCCUCAU 3523 AD-75227 A-150960 UUAUCACACUUAUAGGCCAdTdT 2964 A-150961 UGGCCUAUAAGUGUGAUAAdTdT 3244 UUAUCACACUUAUAGGCCA 3524 AD-75228 A-150962 CAAGUGUGAUAAAUAAACUdTdT 2965 A-150963 AGUUUAUUUAUCACACUUGdTdT 3245 CAAGUGUGAUAAAUAAACU 3525 AD-75229 A-150964 UUACAGACACUGAAUUAAUdTdT 2966 A-150965 AUUAAUUCAGUGUCUGUAAdTdT 3246 UUACAGACACUGAAUUAAU 3526 AD-75230 A-150966 UUUGAAACCAGAAAAUAAUdTdT 2967 A-150967 AUUAUUUUCUGGUUUCAAAdTdT 3247 UUUGAAACCAGAAAAUAAU 3527 AD-75231 A-150968 AUGACUGGCCAUUCGUUAAdTdT 2968 A-150969 UUAACGAAUGGCCAGUCAUdTdT 3248 AUGACUGGCCAUUCGUUAC 3528 AD-75232 A-150970 UUAGUUGAAAAGCAUAUUUdTdT 2969 A-150971 AAAUAUGCUUUUCAACUAAdTdT 3249 UUAGUUGAAAAGCAUAUUU 3529 AD-75233 A-150972 UUUUUAUUAAAUUAAUUCUdTdT 2970 A-150973 AGAAUUAAUUUAAUAAAAAdTdT 3250 UUUUUAUUAAAUUAAUUCU 3530 AD-75234 A-150974 CUGAUUGUAUUUGAAAUUAdTdT 2971 A-150975 UAAUUUCAAAUACAAUCAGdTdT 3251 CUGAUUGUAUUUGAAAUUA 3531 AD-75235 A-150976 UUUGAAAUUAUUAUUCAAUdTdT 2972 A-150977 AUUGAAUAAUAAUUUCAAAdTdT 3252 UUUGAAAUUAUUAUUCAAU 3532 AD-75236 A-150978 UUAUGGCAGAGGAAUAUCAdTdT 2973 A-150979 UGAUAUUCCUCUGCCAUAAdTdT 3253 UUAUGGCAGAGGAAUAUCA 3533 AD-75237 A-150980 UCUAAAAAUGUAACUAAUUdTdT 2974 A-150981 AAUUAGUUACAUUUUUAGAdTdT 3254 UCUAAAAAUGUAACUAAUU 3534 AD-75238 A-150982 UUUACUGUUUAAUAAGCAUdTdT 2975 A-150983 AUGCUUAUUAAACAGUAAAdTdT 3255 UUUACUGUUUAAUAAGCAU 3535 AD-75239 A-150984 UGUCAUAAUAAAAUGGUAUdTdT 2976 A-150985 AUACCAUUUUAUUAUGACAdTdT 3256 UGUCAUAAUAAAAUGGUAU 3536 AD-75240 A-150986 AUAUCUUUCUUUAGUAAUUdTdT 2977 A-150987 AAUUACUAAAGAAAGAUAUdTdT 3257 AUAUCUUUCUUUAGUAAUU 3537 AD-75241 A-150988 UUAGUAAUUACAUUAAAAUdTdT 2978 A-150989 AUUUUAAUGUAAUUACUAAdTdT 3258 UUAGUAAUUACAUUAAAAU 3538 AD-75242 A-150990 AUUAGUCAUGUUUGAUUAAdTdT 2979 A-150991 UUAAUCAAACAUGACUAAUdTdT 3259 AUUAGUCAUGUUUGAUUAA 3539

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments and methods described herein. Such equivalents are intended to be encompassed by the scope of the following claims. 

1. A double stranded ribonucleic acid (dsRNA) agent for inhibiting expression of an insulin-like growth factor binding protein, acid labile subunit (IGFALS) gene forming a double stranded region, wherein the dsRNA agent comprises a sense strand and an antisense strand, wherein said sense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of any one of SEQ ID NO:1, 3, or 5, wherein said antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of any one of SEQ ID NO:2, 4, or 6; and wherein the dsRNA agent comprises at least one modified nucleotide. 2.-4. (canceled)
 5. The dsRNA agent of claim 1, wherein the sense and antisense strands comprise nucleotide sequences selected from the group consisting of any one of the nucleotide sequences in any one of Tables 3, 5, 6, 8, 12, or
 14. 6. (canceled)
 7. A double stranded ribonucleic acid (dsRNA) agent for inhibiting expression of an insulin-like growth factor 1 (IGF-1) gene, wherein said dsRNA agent comprises a sense strand and an antisense strand forming a double stranded region, wherein said sense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of any one of SEQ ID NO:11 or 13, wherein said antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of any one of SEQ ID NO: 12 or 14; and wherein the dsRNA agent comprises at least one modified nucleotide. 8.-10. (canceled)
 11. The dsRNA agent of claim 7, wherein the sense and antisense strands comprise nucleotide sequences selected from the group consisting of any one of the nucleotide sequences in any one of Tables 9, 11, 15, 17, 18, or
 20. 12. (canceled)
 13. The dsRNA agent of claim 1 or 7, wherein substantially all of the nucleotides of said sense strand or substantially all of the nucleotides of said antisense strand comprise a nucleotide modification. 14.-21. (canceled)
 22. The dsRNA agent of claim 1 or 7, wherein at least one of said modified nucleotides comprises a nucleotide modification selected from the group consisting of a deoxy-nucleotide, a 3′-terminal deoxy-thymine (dT) nucleotide, a 2′-O-methyl modified nucleotide, a 2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an unlocked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2′-amino-modified nucleotide, a 2′-O-allyl-modified nucleotide, 2′-C-alkyl-modified nucleotide, 2′-hydroxly-modified nucleotide, a 2′-methoxyethyl modified nucleotide, a 2′-O-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, a non-natural base comprising nucleotide, a tetrahydropyran modified nucleotide, a 1,5-anhydrohexitol modified nucleotide, a cyclohexenyl modified nucleotide, a nucleotide comprising a phosphorothioate group, a nucleotide comprising a methylphosphonate group, a nucleotide comprising a 5′-phosphate, and a nucleotide comprising a 5′-phosphate mimic. 23.-29. (canceled)
 30. The dsRNA agent of claim 1 or 7, further comprising a ligand.
 31. (canceled)
 32. The dsRNA agent of claim 30, wherein the ligand is an N-acetylgalactosamine (GalNAc) derivative.
 33. The dsRNA agent of claim 32, wherein the ligand is

34.-56. (canceled)
 57. The dsRNA agent of claim 1 or 7, wherein each strand has 15-30 nucleotides. 58.-64. (canceled)
 65. The dsRNA agent of claim 1 or 7, wherein said agent further comprises at least one phosphorothioate or methylphosphonate internucleotide linkage. 66.-100. (canceled)
 101. An isolated cell containing the dsRNA agent of claim 1 or
 7. 102. A pharmaceutical composition for inhibiting expression of an IGFALS gene comprising the dsRNA agent of claim
 1. 103. A pharmaceutical composition for inhibiting expression of an IGF-1 gene comprising the dsRNA agent of claim
 7. 104.-106. (canceled)
 107. A method of inhibiting IGFALS expression in a cell, the method comprising: contacting the cell with the dsRNA agent of claim 1; thereby inhibiting expression of the IGFALS gene in the cell.
 108. A method of inhibiting IGF-1 expression in a cell, the method comprising: contacting the cell with the dsRNA agent of claim 7; thereby inhibiting expression of the IGF-1 gene in the cell. 109.-111. (canceled)
 112. A method of treating a subject having a disease or disorder that would benefit from reduction in IGLAS expression or IGF-1 expression, the method comprising administering to the subject a therapeutically effective amount of the dsRNA agent of claim 1 or 7, thereby treating said subject. 113-127. (canceled) 